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Spiro-configured fluorescent probe: Synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging
Accepted Manuscript Title: Spiro-configured fluorescent probe: synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging Author: Haibo Xiao Yanzhen Zhang Shaozhi Li Wu Zhang Zhongying Han Jingjing Tan Shenyao Zhang Jingyan Du PII: DOI: Reference:
S0925-4005(16)30862-0 http://dx.doi.org/doi:10.1016/j.snb.2016.06.010 SNB 20336
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-3-2016 31-5-2016 1-6-2016
Please cite this article as: Haibo Xiao, Yanzhen Zhang, Shaozhi Li, Wu Zhang, Zhongying Han, Jingjing Tan, Shenyao Zhang, Jingyan Du, Spiro-configured fluorescent probe: synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Spiro-configured fluorescent probe: synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging
Haibo Xiao,* Yanzhen Zhang, Shaozhi Li, Wu Zhang, Zhongying Han, Jingjing Tan, Shenyao Zhang, Jingyan Du
Department of Chemistry, Shanghai Normal University, Shanghai 200234, P.R. China
*
1
E-mail:[email protected]
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Highlights
We report a Hg2+ two-photon fluorescent probe based on spirobifluorene for the first time.
The molecule exhibits high selectivity and excellent sensitivity in both UV/Vis absorbance and one-photon fluorescence detection of Hg2+.
The molecule is a pH probe and suitable to detect acidic regions in cells.
The two-photon emission wavelength change before and after addition of Hg2+ is large.
The molecule enables fluorescence imaging of both the probe and mercury ions in cells by two-photon microscopy.
Abstract A novel fluorescent
probe SPF-TSA was prepared by introduction of triphenylamine and dithioacetal
groups on the respective biphenyl branches of
9,9'-spirobifluorene. The probing behaviors toward metal
ions and pH variations were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. SPF-TSA exhibits high sensitivity and selectivity for mercury ions and could be used to detect mercury ions even by naked eye. The detection limit of SPF-TSA for Hg2+ is at least 2.6×10-8 M , which is excellent comparing with other optical sensors for Hg2+. SPF-TSA showes significantly pH-dependent
spectral properties within the acidic pH-range. Especially, the pKa value of SPF-TSA is close to 6.0, which is critical for precisely assessing the acidic environments of some organelles (pH 4.5−6.8). The two-photon absorption cross-section value of SPF-TSA at 800 nm is 248 GM. SPF-TSA emits strong two-photon induced fluorescence and the emission wavelength change before and after addition of Hg2+ was found to be up to140 nm.
Cell imaging studies demonstrate that SPF-TSA enable fluorescence imaging of
both the probe and mercury ions in cells by two-photon microscopy .
Keywords: Probe; Two-photon fluorescence; Mercury ion; Bioimaging; Spirobifluorene; Probe
3
1. Introduction In recent years abundant optoelectronic materials derived from 9,9'-spirobifluorene have been reported [1-2,3]. This class of dye exhibits fascinating characteristics, such as highly rigid molecular structure, increased
stability,
relatively
sharp
fluorescence
emission,
high
extinction
coefficients
and
photoluminescence (PL) quantum yields. Besides, the optoelectronic properties of spirobifluorene deriatives are easy to tune because the two biphenyl branches of
spirobifluorene connected at a quaternary center
through σ-bonds can be independently tailored [1]. Up to nowadays, some
fluorescent probes based on
spirobifluorene have been studied [1-2]. The reported spirobifluorene fluorescent probes, however, usually exhibit absorption and emission in the ultraviolet and visible regions, which can be highly absorbed by biological substances and easily cause damage to biological substances. Thus, their practical utilization in biological systems is limited [4]. Two-photon excited fluorescent microscopy (TPM ), which utilizes two near-infrared photons of lower energy for the excitation, is a powerful method for three-dimensional imaging in biological systems
due to several distinct advantages, such as increased penetration depth, lower tissue
auto-fluorescence and self-absorption [1, 5-10]. Detecting mercury ions has received considerable attention owing to their extremely toxic impact on the environment and human health [11-12]. It is a continuous challenge for researchers to develop effective and sensitive detection techniques for Hg2+ ions. Among the different detection methods, the use of fluorescence techniques for Hg2+ detection has been much appreciated owing to their low detection limit, real-time detection, portability, high selectivity and sensitivity and simple operation procedures [13]. To date, many successful fluorescent chemosensors for Hg2+ detection have been reported, however, only a few have been used in vivo experiments [14]. Chemosensors involving the use of a dithioacetal group as a receptor unit are presented as a new and emerging approach for the detection of Hg2+ ions. The dithioacetal moiety not 4
only serves as a coordination site for Hg2+ ions, but also provides good water solubility and high selectivity to the sensor [14]. Up to the present, few papers have reported the two-photon fluorescence detection of Hg2+ [5, 13, 15, 16], because of the limitations in constructing two-photon fluorescent probes for Hg2+ with high selectivity and sensitivity [15].
Much effort has been made toward development of optical pH probes because of the importance of pH measurements in various scientific research areas and applications[17-19]. Intracellular pH
is an essential
factor that controls many cellular metabolic processes, such as signaling, endocytosis, apoptosis, and proliferation. Some organelles, such as lysosomes (pH 4.0−5.5) and endosomes (pH 4.5−6.8), have acidic pH values, which is linked to enzyme activity and protein degradation. For precisely assessing the acidic environments, there is a critical need for the pH probes with pKa values in the range of 4.5-6.5 [18,19]. We
have
reported
previously
the
strong
two-photon
induced
fluorescence
property
of
2,7-bis-(4-(N,N-diphenylamino) phen-1-yl)-9,9'-spirobifluorene (SPF-TP, Scheme 1) [20]. Herein, a novel chromophore, namely, 2,2',2'',2'''-(((2',7'-bis(4-(diphenylamino)phenyl)-9,9'-spirobi[fluorene]-2,7-diyl) bis(methanetriyl))tetrakis(sulfanediyl))tetraacetic acid
(SPF-TSA)
was obtained (Scheme 2).
design of the target molecule, the dithioacetal groups were introduced to serve as
Hg2+
In the
receptor units.
The triphenylamine -containing conjugated branch acts as the two-photon fluorophore. The probing behaviors of SPF-TSA toward metal ions, proton and the applications in two-photon fluorescence biological imaging were investigated.
2. Experimental section 2.1 Materials and Reagents. Compound 2',7'-dibromo-9,9'-spirobi[fluorene]-2,7-dicarbaldehyde (1) was prepared according to our previous method [2, 21]. All chemicals were commercially available and of analytical grade. Solutions 5
of metal ions were all prepared from their chloride salts. Ultrapure water was used from a Millipore water purification system.
2.2. Instruments and Measurements. Melting points were determined with an XT-4A apparatus and are uncorrected. 1H NMR and 13C NMR spectra were obtained on a Bruker DRX 400 MHz spectrometer. Chemical shifts were reported in ppm relative to a Me4Si standard. Steady-state emission spectra were recorded on Perkin Elmer LS55 instrument. Visible absorption spectra were determined on Perkin Elmer Lambda 35 spectrophotometer. Two-photon absorption cross-section was measured by the two-photon excited fluorescence (TPEF) technique using a Ti:Sapphire laser (Spectra-Physics). This laser provided pulses of 100 fs of duration with repetition rate of 80MHz at wavelength of 800 nm. The laser beam was focused into a quartz cell of 1cm path length by using a 5 cm focal-length lens. A half-wave plate and a polarizer were used to control the excitation intensity. The induced two- photon fluorescence was collimated by a lens at a direction perpendicular to the pump beam. To minimize the attenuation of fluorescence due to linear absorption effects, the excitation beam was focused as close as possible to the lateral wall of the quartz cell. The TPEF was then focused into the input slit of an imaging spectrograph and recorded at the exit with a CCD camera. To calculate the TPEF cross sections, Rhodamine B in methanol solution (10 mM) was utilized as references for the calculation. All the samples and standards were tested under the same experimental conditions. The TPEF cross-sections were calculated by the equation:σTPEF=σref cref nref F/cnFref, where c and n were the concentration and refractive index of the samples and reference, and F was the integral of the TPEF spectrum. The two photon cross-section σ was then calculated by the equation σ =σTPEF/Φ, where Φ was the fluorescence quantum yield.
6
2.3 Synthesis of SPF-TSA 2.3.1
Preparation of compound 2
To a solution of 1(52.8mg, 0.1mmol) and methyl thioglycolate (45μl, 1mmol) in dry dichloromethane (2 ml), BF3.Et2O (65μl, mmol) was added. After stirring at 0℃ for 2 h, the reaction was quenched by addition of saturated NaHCO3. The mixture was extracted with dichloromethane (10 ml×3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using petroleum ether/ethyl acetate/dichloromethane (80:1:4-20:5:3,v/v) to yield 60 mg
of 2 as white solid (65.4%). Mp:143~145℃. 1H NMR (400 MHz,
CDCl3) δ 7.81 (d, J = 8.0 Hz, 1H ), 7.69 (d, J = 8.0 Hz,1H ), 7.56-7.48 (m, 2H ),6.80 (d, J = 1.6 Hz, 1H ), 6.72 (s, 2H ), 5.17 (s, 1H ), 3.60 (s, 6H ), 3.35 (d, J = 12.0 Hz, 2H ), 3.08 (d, J = 16.0 Hz, 2H ). 13C NMR (101 MHz, CDCl3) δ 170.38, 149.88, 148.15, 141.51, 139.93, 138.95, 131.65, 128.58, 127.42, 123.75, 122.23, 121.90, 121.01, 65.68, 53.71, 52.64, 33.86.
2.3.2
Preparation of compound 3
A mixture of compound 2 (300mg, 0.327mmol), 4-(diphenylamino)phenylboronic acid (207.8mg, 0.72 mmol), Pd(OAc)2 (4mg, 0.017 mmol), PPh3 (16.7mg, 0.068 mmol) and Na2CO3 (148mg,1.32mmol) in dry THF (20 ml) were reacted under nitrogen atmosphere. After the mixture was refluxed at 80℃ for 18 h, the mixture was cooled to room temperature. The solvent was evaporated and the residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate:dichloromethane = 50:1:3-30:5:6, V/V) to afford yellowish solid (300mg, 73.7 %). Mp:80-82℃. 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.0 Hz, 2H ), 7.81 (d, J = 8.0 Hz, 2H ), 7.60 (dd, J = 8.0, 1.6 Hz, 2H ), 7.51 (dd, J = 8.0, 1.6 Hz, 2H ), 7.28 (d, J = 8.0
7
Hz, 4H ), 7.22 (t, J = 8.0 Hz, 8H ), 7.10-7.05 (m, 8H ), 7.04-6.95 (m, 8H ), 6.85 (d, J = 1.2 Hz, 2H ), 6.79 (d, J = 1.2 Hz, 2H ), 5.14 (s, 2H ), 3.52 (s, 12H ), 3.32 (d, J = 16.0 Hz, 4H ), 3.05 (d, J = 16.0 Hz, 4H ).
2.3.3
Preparation of SPF-TSA
A mixture of 3 (26mg, 0.02 mmol) and aq. NaOH (0.15 ml, 1 N) in methanol (3 ml) was stirred for 10 h at rt. Then, pH of the solution was neutralized by aq. HCl (1 M). Na2CO3 was added to the solution to obtain sodium salt of SPF-TSA. Then the mixture extracted with ethylacetate (20 ml×3) for purification. The aqueous phase then was neutralized again with aq. HCl and extracted with ethylacetate. The extract was dried over Na2SO4. The solvent was removed at reduced pressure to afford SPF-TSA with 42% yield as a yellowish solid. Mp:172-173℃. 1H NMR (400 MHz, DMSO): δ 12.51 (s, 4H ), 8.11 (d, J = 8.0 Hz, 2H ), 8.03 (d, J = 8.0 Hz, 2H ), 7.70 (d, J = 8.0 Hz, 2H ), 7.46 (d, J = 8.0 Hz, 2H ), 7.37 (d, J = 8.0 Hz, 4H ), 7.27 (t, J = 8.0 Hz, 8H ), 7.05-6.95 (m, 12H ), 6.91 (m, J = 12.0 Hz, 4H ), 6.81 (s, 2H ), 6.70 (s, 2H ), 5.19 (s, 2H ), 3.25 (d, J = 16.0 Hz, 4H ), 3.12 (d, J = 16.0 Hz, 4H ). 13C NMR (101 MHz, CDCl3) δ 176.91, 150.09, 149.14, 147.79, 147.45, 141.96, 140.83, 140.61, 138.56, 135.07, 129.50, 128.44, 127.94, 127.14, 124.62, 124.00, 123.93, 123.20, 122.26, 120.93, 120.86, 66.37, 52.46, 33.50. Elemental analysis (%) calcd for C71H54N2O8S4: C 71.57, H 4.57, N 2.35, O 10.74, S 10.76. Found: C 91.00, H 6.37, N 3.05. TOF MS ( ES+ ): 1190. 3.
2.4. Confocal Imaging in Live Cells. 293T cells were grown in Dulbecco’s modified Eagle’s (DMEM) containing 4500 mg/L glucose, 3.7 g/L NaHCO3, 2 units/mlnystatin, 50 μg/ml gentamicin, 10 mM Hepes, and 10% fetal bovine serum at 37℃ in a humidified atmosphere containing 5% CO2. Two groups of cells were exposed to dye (50 μM) for 30 min and then washed three times with PBS buffer to remove excess probe, and one group of cells were incubated 8
with Hg2+ (100 μM) for 30 min and then washed with PBS buffer to remove unreacted Hg2+. TPEF images were taken on an Ultimal IV confocal microscope equipped with a femtosecond Ti:sapphire laser.
3. Results and discussion 3.1. Linear absorption and one-photon-excited fluorescence
The linear spectroscopic properties of SPF-TSA were measured in five organic solvents of different polarities with the concentration of 1.0× 10-5 mol L-1 (Table 1). It can be seen from Fig.1 and Table 1, SPF-TSA and SPF-TP have very similar absorption and emission spectral properties. The spectral features of
SPF-TSA were influenced only slightly by dithioacetal moiety.
The absorbance bands of
SPF-TSA
and SPF-TP located at the high-energy side with peaks around 300nm are mainly attributed to n-π* transitions derived from the triphenylamine unit [22], whereas the low-energy bands around 328-415 nm result from the π-π* transition of the triphenylamine-capped biphenyl branches [23]. The absorption spectra of SPF-TSA present a small shoulder peak at about 320 nm ( Fig.1), which can be attributed to the dithioacetal moiety [16, 24]. The PL emission spectra
of SPF-TSA
show significant red-shifts from
low-polarity to high-polarity solvents along with decreased PL intensity (Table 1), which indicates that the compound has a highly charged excited state due to photo induced charge transfer [25] .
3.2 Selective recognition of Hg2+
The sensing ability of SPF-TSA for metal cations (Li+, Na+, Co2+, Cu2+, Ca2+, Cd2+, Ag+, Zn2+, Hg2+, Pb2+, Mg2+, Mn2+, Ni2+, Fe3+) were investigated. When 2.0× 10-5M of Hg2+ ions were added to SPF-TSA in DMSO/water (1:1, V/V)
solution (1.0× 10-5M), the absorption and fluorescence spectra underwent
dramatic changes and the values of the molar absorptivity and the relative fluorescence intensity were decreased (Fig.2). The absorption bands at 370 nm decreased with simultaneous disappearance of the small 9
shoulder peaks at about 320 nm, generating an isosbestic point at 395 nm (Fig. 2a, Fig.S1). According to the literature [14,16,24],
SPF-TSA
underwent a mercury-promoted hydrolysis as shown in Scheme 3,
giving the product SPF-DA at room temperature. The sensing mechanism of SPF-TSA is confirmed by the NMR data of SPF-DA (Fig. S2 and Fig.S3), which were recorded after reaction. Further evidence for the formation of
the hydrolysis product
SPF-DA
was provided by the appearance of an isosbestic point in
the absorption spectra of SPF-TSA upon addition of Hg2+ (Fig. 2a, Fig.S1).
Along with the addition of
Hg2+, a color change from yellowish to colorless was observed (Fig.2a, inset), which suggested that SPF-TSA could serve as a “naked-eye” sensor for the detection of
Hg2+.
As depicted in Fig. 2a, Ag+,
Pb2+ and Ni2+ had a slight effect on the absorption of SPF-TSA. Upon addition of Hg2+, distinct fluorescent quenching can be observed
under the UV light (λ = 365 nm) (Fig.2b, inset). The fluorescence emission
change, formed upon addition of different concentration of Hg2+ to a solution of SPF-TSA (1.0× 10-5M), is shown in Figure 3. The fluorescence became weak gradually and blue-shifted with the increase of concentration. The fluorescence
the Hg2+
ratios (Io– I) / Io toward the surveyed metal ions are displayed in Fig.4.
The results show highly selective response of receptor SPF-TSA to Hg2+ as compared to the other metal ions −
−
(10-5M, 1:1, V/V) as a function of Hg2+ concentration in the range of 1 ×10 6 -1×10 5 M. [26]. A good linearity between Io / I and concentration of Hg2+ in the range of 1 ×10−6 -1×10−5 mol/L was obtained with a linearly dependent coefficient R2 of 0.992 (Fig. 5). Hence, a quantitative measurement of [Hg2+] was possible in DMSO/water (1:1, V/V)
in this concentration range [13]. The detection limit (DL)
was calculated by the following equation: DL = 3σ s/ms, where σs is the standard deviation of
blank sample
and ms is the slope of calibration curve. The detection limit of SPF-TSA was at least 2.6×10-8M, which is better than many reported data [26-30].
3.3. pH-dependent spectral properties For the analysis of pH-dependent properties, the spectra 10
of
SPF-TSA were recorded in DMSO-water
mixture (1:1, v/v, 1.0× 10-5M) between pH 1.01 and 13.68 using HCl and NaOH for pH adjustment (Fig. 6, Fig.7). In alkaline media (pH˃7.65), SPF-TSA showed no interesting responses toward pH variations. Therefore, the following studies were restricted to the survey of the spectral behavior of
SPF-TSA in
acidic condition. It can be seen from Fig. 6, with decreased pH, the absorption bands of SPF-TSA become weak and the low-energy band at 366 nm is red shifted gradually. When the pH value reached 1.01, the low-energy absorption band of SPF-TSA SPF-TSA
appeared at around 386 nm. In contrast, the fluorescence spectra of
are characterized by hyperchromic shifts with the decrease of the pH (Fig.7). When the pH is
decreased from 6.20 to 5.40, the fluorescence emission intensities of SPF-TSA decreased significantly. An anaysis of
F470 versus pH was shown in Fig.7 (inset) and the pKa value of
SPF-TSA determined by
fluorescence titration plots was found to be comparable at ∼6.00 [27,31,32]. This result
indicates that
SPF-TSA is suitable to detect acidic regions in cells and tissues. The pH-dependent spectral properties of SPF-TSA may be due to the protonation of
the nitrogen atom of
triphenylamine moiety under acidic
condition. It has been pointed out that the sensing procedure of Hg2+ is usually affected by solution pH value [33-35]. The emission intensities of
SPF-TSA with and without Hg2+ in phosphate buffered saline under various
pH values are shown in Fig. 8. There was no dramatic spectral change for SPF-TSA at higher than pH 8. From the view of sensitivity, the suitable range of pH for Hg2+ determination is found to be within 8-11 [35-38]. The sensing operations can be carried out in buffer solutions so that the influence from various pH values can be avoided [33].
3.4.Two-photon absorption cross-section and two-photon fluorescence
11
cell imaging
The two-photon cross-section values were measured by two-photon excited fluorescence (TPEF) technique in THF at a concentration of 1.0 ×10-5mol L-1 [31]. The values are 248GM (1 GM= 1×10-50cm4 s per photon) for SPF-TSA and 686GM for SPF-DA at 800 nm, respectively. SPF-TSA emits blue up-converted fluorescence with the maximal peak at about 435 nm and the profile is similar to that of the single-photon induced fluorescent spectrum (Fig.1, Fig.9a). However, the two-photon induced emission spectrum of SPF-DA in THF is different from that data obtained by one-photon excitation in the same solvent (Fig.9a, Fig.S4). Two emission bands at 429nm and 556nm were observed for SPF-DA on one-photon excitation
in THF (Fig.S4). It can be seen from Fig. 9b, SPF-DA exhibited only one emission
band located at ca. 575nm on 800nm laser excitation [32].
The result could be explained as follows (i) The
two-photon induced emission band located at 575nm may be the ICT (intramolecular charge-transfer) emission band. This viewpoint can be supported by the molecular structure and one-photon spectra of
fluorescence
SPF-DA. From the point of view of molecular structure, SPF-DA is a spiro-configured donor
(D)-acceptor (A) compound, in which electron-rich triphenylamine
and electro-deficient formyl moieties
are present on the two respective biphenyl branches of a 9,9'-spirobifluorene core [39]. The strong electron-withdrawing effect of the formyl group is beneficial for promoting interaction between the donor and acceptor parts in the excited state [32, 40]. Such spirobifluorene-bridged bipolar systems usually exhibit an efficient photoinduced excitation characteristic of a ICT emission band [32, 40]. As depicted in Fig.S4, the emission band at ca.525nm on single-photon excitation showed significant red-shifts from low-polarity to high-polarity solvents along with decreased PL intensity, supporting the assignment of
it to ICT
process [25]. The photo induced charge transfer would be happened in the same way for two- photon excitation and the ICT band (at 575nm) increases significantly due to the intense laser, while the normal emission band maximized at about 450nm was too weak to be 12
observed (Fig.9b) [32]. Or (ii) The large red
shift of the two-photon emission of SPF-DA may be due to some structural change of the molecule by high laser intensity in the two- photon absorption event. The two-photon showed about 140nm red-shifted as compared to that
induced emission of
SPF-DA
of SPF-TSA. The emission wavelength change
(140-nm shift) before and after addition of Hg2+ is larger than the reported two-photon fluorescent probes [16]. The TPEF intensity of SPF-TSA was decreased upon the addition of mercury ion and acidification,
respectively (Fig.10 and Fig.11). The results suggest that SPF-TSA can be used as an efficient two-photon fluorescence probe for the detection of Hg2+ and proton [41].
The pH values of most biological samples are
within the range 5.25-8.93 [36]. The sensitivity of
SPF-TSA to pH variations is detrimental to the detection of Hg2+ in living cells. Fortunately, SPF-TSA is
a reaction-based two-photon probe
for mercury species. Sensing of Hg2+ ions in cells by two-photon
microscopy can be achieved via a emission change with a significant wavelength shift. Thus, we have demonstrated the feasibility of the application of SPF-TSA to the two-photon fluorescence imaging of Hg2+ present in live
cells [14, 42]. Two groups of 293T cells were exposed to dye (50μM) for 30 min and
then washed three times with PBS buffer to remove excess dye, and one group of cells were incubated with Hg2+ (100μM) for 30 min and then washed with PBS buffer to remove unreacted Hg2+. The cells treated with the dye alone show bright blue fluorescence by two-photon microscopy (Fig.12, left). The cells treated with Hg2+ show yellow-green fluorescence owing to the hydrolysis product SPF-DA (Fig. 12, right). The results demonstrate that SPF-TSA enables fluorescence imaging of both the probe and Hg2+ in cells. The cytotoxic effect of SPF-TSA against 293T cells was assessed by MTT assay over a 24 h period. 293T cells incubated with 2.5 mM of SPF-TSA remained 90% viable after 24 h of feeding time (In contrast, 100% survival is observed for 293T cells not treated with SPF-TSA). The result suggests that SPF-TSA exhibits low toxicity 13
to 293T cells in our measuring range and could be used to detect Hg2+ in vivo with little damage [13,43].
4. Conclusions
We have developed a spirobifluorene-based fluorescence probe. In comparison to the reported results, SPF-TSA is distinguished by exclusive selectivity and sensitivity for mercury species, large two- photon absorption cross-section , and large emission wavelength change (140-nm shift) before and after addition of Hg2+ . SPF-TSA enable fluorescent imaging of both the probe and mercury ions in cells by two-photon microscopy . In addition,
SPF-TSA posseses excellent indicator properties within the acidic
pH-range. The results demonstrated that spirobifluorene is a suitable fluorophore core for highly sensitive two-photon fluorescent probes.
Acknowledgments
This work was financially supported by the Natural Science Foundation of Shanghai City of China (No.15ZR1431400).
Appendix A. Supplementary data Supplementary data related to this article can be found at
14
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[15] F. Liu, C.Q. Ding, M. Jin, Y. Tian, A highly selective two-photon fluorescent probe for the determination of mercury ions. Analyst. 140(2015) 3285-3289. [16] A. S. Rao, D. Kim, T.J. Wang, K. H. Kim, S. Hwang, Reaction-based two-photon probes for mercury ions: fluorescence imaging with dual optical windows. Org.Lett. 14(2012) 2598-2601. [17] J.L. Fan , C.Y. Lin, H.L. Li , P. Zhan, J.Y. Wang , S. Cui, M.M. Hu , G.H. Cheng , X.J. Peng, A ratiometric lysosomal pH chemosensor based on fluorescence resonance energy transfer . Dyes Pigm. 99 (2013) 620-626. [18] J.Y. Han, K. Burgess, Fluorescent indicators for intracellular pH. Chem. Rev. 110(2010) 2709-2728. [19] H.J. Kim, C.H. Heo, H.M. Kim, Benzimidazole-based ratiometric two-photon fluorescent probes for acidic pH in live cells and tissues. J. Am. Chem. Soc. 135(2013) 17969-17977.
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[20] H.Y. Yin, H.B. Xiao, L. Ding, C. Zhang, A.M. Ren, B. Li. A bistriphenylamine-substituted spirobifluorene derivative exhibiting excellent nonlinearity/transparency/thermal stability trade-off and strong two-photon induced blue fluorescence. Mater. Chem. Phys. 151(2015)181-186. [21] H.B. Xiao, H.Y. Yin, X.Y. Zhang. Improved nonlinearity transparency thermal stability trade-off with spirobifluorene-bridged donor-π-acceptor chromophores. Org. Lett.14(2012)5282-5285. [22] F. Polo, F. Rizzo, M. eiga-Gutierrez, L. De Cola, S. Quici, Efficient greenish blue electrochemiluminescence from fluorine and spirobifluorene derivatives. J.Am.Chem.Soc. 134(2012) 15402-15409. [23] S.-Y. Ku, W.-Y. Hung, C.-W. Chen, S.-W. Yang, E. Mondal,
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J. Jiang ,A highly selective fluorescent and colorimetric
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16
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17
Biographies Haibo Xiao received his PhD in chemistry from East China University of Science and Technology in 2005. He is currently an associate professor of the chemistry department at Shanghai Normal University. His main research fields are chemical sensors, organic nonlinear optical materials and organic light-emiting material. Yanzhen Zhang is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Shaozhi Li is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Wu Zhang is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Zhongying Han is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Jingjing Tan is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Shenyao Zhang is a student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Jingyan Du is a student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors.
18
SPF-TSA SPF-TP
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 300
350
400 450 Wavelength (nm)
500
550
Emission Intensity (arb.unit.)
Absorbance
1.0
0.0
Fig. 1. Normalized one-photon absorption and fluorescent emission of SPF-TSA and SPF-TP in THF.
19
0.8 Hg
0.7
Absorbance
0.6 0.5
blank + Ag 2+ Ca 2+ Cd 2+ Co 2+ Hg + Li 2+ Mg 2+ Mn + Na 2+ Ni 2+ Pb 2+ Zn 3+ Fe 2+ Cu
2+
a)
0.4 0.3 0.2 0.1 0.0 325
350
375 400 Wavelength(nm)
425
450
Fig.2 (a)The absorption spectra of SPF-TSA upon addition of various metal ions in DMSO/water (1:1, V/V) solution (1.0× 10-5M). Inset: the photograph
of SPF-TSA solution (1.0× 10-5M) in the presence of
2.0× 10-5M of Hg2+. (b)The fluorescence spectra of SPF-TSA upon addition of various metal ions in DMSO/water (1:1, V/V) solution (1.0× 10-5M). -5
-5
10 M) in the presence of 2.0× 10 M
20
2+
Inset: the photograph
Hg under the UV light.
of SPF-TSA solution
(1.0×
blank 1×10-7 5×10-7 1×10-6 2×10-6 4×10-6 6×10-6 8×10-6 1×10-5 2×10-5 4×10-5 6×10-5 8×10-5 1×10-4 2×10-4
Fluorescence Intensity
1600
1200
800
400
0 400
450
500
550
600
Wavelength(nm)
Fig.3. Fluorescence emission spectra of SPF-TSA(1.0× 10-5M) upon addition of
21
Hg2+ in
various concentrations.
0.8
(I0-I)/I0
0.6
0.4
0.2
0.0
Blank Ag[I] Ca[II]Cd[II]Co[II]Hg[II] Li[I] Mg[II]Mn[II] Na[I] Ni[II] Pb[II] Zn[II]Fe[III]Cu[II]
Fig.4. Fluorescence ratio (Io– I) / Io of receptor SPF-TSA upon addition of a particular metal salt in DMSO/water (1:1, V/V, 1.0× 10-5M ).
22
20 5
2+
18
Y = 1.598x10 [Hg ]+ 3.10459
16
R =0.9922
2
I0/I
14 12 10 8 6 4 0
200
400
600 2+
800
1000
-8
[Hg ]/10 M
Fig.5. The relative fluorescence intensity (I0/I) of SPF-TSA
23
DMSO/water solution
0.55
pH=7.65
0.50 0.45
Absorbance
0.40 0.35 0.30 0.25
pH=1.0
0.20
1.01 1.50 2.02 3.01 3.98 5.00 6.20 6.96 7.65
0.15 0.10 0.05 0.00 300
350 400 450 Wavelength(nm)
500
550
Fig. 6. Absorption spectra of SPF-TSA (1.0 ×10-5M) under acidic conditions.
24
2000
2000 1800
pH=7.65
1600
Intensity
1400 1200 1000
1.01 1.50 2.02 2.55 3.01 3.98 4.20 5.00 5.40 6.20 6.96 7.65
Fluorescence Intensity
800
1500
600 400 200 2
4
6
8
10
12
14
pH
1000
500
pH=1.01
0 400
450
500
550
600
Wavelength(nm)
Fig. 7. One-photon-excited fluorescence spectra of SPF-TSA (1.0 ×10-5M)
25
under acidic conditions..
0
2
4
6
8
10 10
2000 SPF-TSA SPF-TSA+Hg
Emission Intensity (a.u.)
1800
8
1600 1400
6
1200 1000
4
800 600
2
400 200 0
0
1
2
3
4
5
6
7
8
9
10
11
12
pH
Fig. 8. Luminescence intensity of SPF-TSA (1.0 ×10-5M) at 470 nm in the absence and presence of 5 equiv of Hg2+ as a function of pH. Conditions: λex 360 nm.
26
3500 2
4
6
8
10
0
10
4
3.6 2
3.4 3.2 1.6
6000 4000
0
1.7
1.8
1.9
2.0
2.1
2.2
logI 0
300mw 250mw 200mw 150mw 100mw 50mw
a)
2000
400
450
500
Wavelength(nm)
550
600
2500
6
8
10 10
200mw 250mw 340mw 410mw 480mw
8
2.78
3000
Fluorescence Intensity
3.8
4
Experimetal data Slope=0.20
8
6
8000
2
2.80
Experimental data Slope=1.89
4.0
logIF
Fluorescence Intensity
0
4.2
6
logIF
4.4
10000
2.76 4
2.74
2
2.72 2.70 2.0
0
2.1
2.2
2.3
2.4
2.5
logI0
2000 1500
b)
1000 400
450
500
550
600
650
700
750
Wavelenth(nm)
Fig.9.Two-photon excited fluorescence spectra of SPF-TSA (a) and SPF-DA (b) under different pumped powers at 800 nm. Inset, power dependence ofthe TPA-induced up conversion emission intensity on the input intensity.
27
Fluorescence intensity（ Counts）
0
2
4
6
8
blank 4.0×10-6 8.0×10-6 1.5×10-5 2.0×10-5 2.2×10-5 2.4×10-5 2.6×10-5 2.8×10-5
5000 4000 3000 2000
10 10
8
6
4
2
1000 0
0
400
450
500
550
600
Wavelength(nm)
Fig.10. The two-photon fluorescence spectra of SPF-TSA (1.0 ×10-5M ) in DMSO/water (1:1,v/v) upon the addition of mercury ions (λex= 800 nm).
28
0
2
4
6
8
10 10
5000
Fluorescence Intensity
pH=7.03
7.03 6.90 6.25 5.41 4.61 3.72 2.87 2.01
4000
3000
2000 pH=2.01
1000
0
8
6
4
2
0
450
500
550
600
Wavelenth (nm)
Fig.11. The two-photon fluorescence spectra of SPF-TSA (1.0 ×10-5M ) in DMSO/water (1:1,v/v) under acidic conditions (λex= 800 nm).
29
Fig. 12.Two-photon
microscopy images of 293T cells labeled with SPF-TSA before (left)
and after (right) addition of
30
Hg2+ (100 μM). Excitation at 800 nm.
Scheme 1. The structures of SPF-TP.
31
Scheme 2. The synthesis route of
32
SPF-TSA.
Scheme 3. Mercury ion-promoted hydrolysis of
33
SPF-TSA.
Table 1. Linear Optical Properties of solvent
λabs (nm)
λfl(nm) a
Φb
Toluene
296, 370
426
0.88
THF
296, 368
428
0.85
CH2Cl2
295, 372
441
0.81
DMF
296, 374
454
0.70
Toluene
301, 372
418
0.98
THF
297, 369
425
0.90
CH2Cl2
296, 369
438
0.87
DMF
298, 373
458
0.70
compound
SPF-TSA
SPF-TP
a
excited at 370 nm.
b
φ Fluorescence quantum yield, was dtermined by using Rhodamine B as the standard.
34
SPF-TSA and SPF-TP.
S0925-4005(16)30862-0 http://dx.doi.org/doi:10.1016/j.snb.2016.06.010 SNB 20336
To appear in:
Sensors and Actuators B
Received date: Revised date: Accepted date:
23-3-2016 31-5-2016 1-6-2016
Please cite this article as: Haibo Xiao, Yanzhen Zhang, Shaozhi Li, Wu Zhang, Zhongying Han, Jingjing Tan, Shenyao Zhang, Jingyan Du, Spiro-configured fluorescent probe: synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging, Sensors and Actuators B: Chemical http://dx.doi.org/10.1016/j.snb.2016.06.010 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Spiro-configured fluorescent probe: synthesis and applications in the determinations of Hg2+ and proton, and two-photon fluorescence imaging
Haibo Xiao,* Yanzhen Zhang, Shaozhi Li, Wu Zhang, Zhongying Han, Jingjing Tan, Shenyao Zhang, Jingyan Du
Department of Chemistry, Shanghai Normal University, Shanghai 200234, P.R. China
*
1
E-mail:[email protected]
Graphical
abstract 2000
2000 1800
pH=7.65
1600
Intensity
1400 1200 1000
1.01 1.50 2.02 2.55 3.01 3.98 4.20 5.00 5.40 6.20 6.96 7.65
Fluorescence Intensity
800
1500
600 400 200 2
4
6
(I0-I)/I0
0.2
0.0
2
Blank Ag[I] Ca[II]Cd[II]Co[II]Hg[II] Li[I] Mg[II]Mn[II] Na[I] Ni[II] Pb[II] Zn[II]Fe[III]Cu[II]
14
pH=1.01
450
500 Wavelength(nm)
0.4
12
500
400
0.6
10
1000
0
0.8
8
pH
550
600
Highlights
We report a Hg2+ two-photon fluorescent probe based on spirobifluorene for the first time.
The molecule exhibits high selectivity and excellent sensitivity in both UV/Vis absorbance and one-photon fluorescence detection of Hg2+.
The molecule is a pH probe and suitable to detect acidic regions in cells.
The two-photon emission wavelength change before and after addition of Hg2+ is large.
The molecule enables fluorescence imaging of both the probe and mercury ions in cells by two-photon microscopy.
Abstract A novel fluorescent
probe SPF-TSA was prepared by introduction of triphenylamine and dithioacetal
groups on the respective biphenyl branches of
9,9'-spirobifluorene. The probing behaviors toward metal
ions and pH variations were investigated via UV/Vis absorption spectra as well as one-photon fluorescence changes. SPF-TSA exhibits high sensitivity and selectivity for mercury ions and could be used to detect mercury ions even by naked eye. The detection limit of SPF-TSA for Hg2+ is at least 2.6×10-8 M , which is excellent comparing with other optical sensors for Hg2+. SPF-TSA showes significantly pH-dependent
spectral properties within the acidic pH-range. Especially, the pKa value of SPF-TSA is close to 6.0, which is critical for precisely assessing the acidic environments of some organelles (pH 4.5−6.8). The two-photon absorption cross-section value of SPF-TSA at 800 nm is 248 GM. SPF-TSA emits strong two-photon induced fluorescence and the emission wavelength change before and after addition of Hg2+ was found to be up to140 nm.
Cell imaging studies demonstrate that SPF-TSA enable fluorescence imaging of
both the probe and mercury ions in cells by two-photon microscopy .
Keywords: Probe; Two-photon fluorescence; Mercury ion; Bioimaging; Spirobifluorene; Probe
3
1. Introduction In recent years abundant optoelectronic materials derived from 9,9'-spirobifluorene have been reported [1-2,3]. This class of dye exhibits fascinating characteristics, such as highly rigid molecular structure, increased
stability,
relatively
sharp
fluorescence
emission,
high
extinction
coefficients
and
photoluminescence (PL) quantum yields. Besides, the optoelectronic properties of spirobifluorene deriatives are easy to tune because the two biphenyl branches of
spirobifluorene connected at a quaternary center
through σ-bonds can be independently tailored [1]. Up to nowadays, some
fluorescent probes based on
spirobifluorene have been studied [1-2]. The reported spirobifluorene fluorescent probes, however, usually exhibit absorption and emission in the ultraviolet and visible regions, which can be highly absorbed by biological substances and easily cause damage to biological substances. Thus, their practical utilization in biological systems is limited [4]. Two-photon excited fluorescent microscopy (TPM ), which utilizes two near-infrared photons of lower energy for the excitation, is a powerful method for three-dimensional imaging in biological systems
due to several distinct advantages, such as increased penetration depth, lower tissue
auto-fluorescence and self-absorption [1, 5-10]. Detecting mercury ions has received considerable attention owing to their extremely toxic impact on the environment and human health [11-12]. It is a continuous challenge for researchers to develop effective and sensitive detection techniques for Hg2+ ions. Among the different detection methods, the use of fluorescence techniques for Hg2+ detection has been much appreciated owing to their low detection limit, real-time detection, portability, high selectivity and sensitivity and simple operation procedures [13]. To date, many successful fluorescent chemosensors for Hg2+ detection have been reported, however, only a few have been used in vivo experiments [14]. Chemosensors involving the use of a dithioacetal group as a receptor unit are presented as a new and emerging approach for the detection of Hg2+ ions. The dithioacetal moiety not 4
only serves as a coordination site for Hg2+ ions, but also provides good water solubility and high selectivity to the sensor [14]. Up to the present, few papers have reported the two-photon fluorescence detection of Hg2+ [5, 13, 15, 16], because of the limitations in constructing two-photon fluorescent probes for Hg2+ with high selectivity and sensitivity [15].
Much effort has been made toward development of optical pH probes because of the importance of pH measurements in various scientific research areas and applications[17-19]. Intracellular pH
is an essential
factor that controls many cellular metabolic processes, such as signaling, endocytosis, apoptosis, and proliferation. Some organelles, such as lysosomes (pH 4.0−5.5) and endosomes (pH 4.5−6.8), have acidic pH values, which is linked to enzyme activity and protein degradation. For precisely assessing the acidic environments, there is a critical need for the pH probes with pKa values in the range of 4.5-6.5 [18,19]. We
have
reported
previously
the
strong
two-photon
induced
fluorescence
property
of
2,7-bis-(4-(N,N-diphenylamino) phen-1-yl)-9,9'-spirobifluorene (SPF-TP, Scheme 1) [20]. Herein, a novel chromophore, namely, 2,2',2'',2'''-(((2',7'-bis(4-(diphenylamino)phenyl)-9,9'-spirobi[fluorene]-2,7-diyl) bis(methanetriyl))tetrakis(sulfanediyl))tetraacetic acid
(SPF-TSA)
was obtained (Scheme 2).
design of the target molecule, the dithioacetal groups were introduced to serve as
Hg2+
In the
receptor units.
The triphenylamine -containing conjugated branch acts as the two-photon fluorophore. The probing behaviors of SPF-TSA toward metal ions, proton and the applications in two-photon fluorescence biological imaging were investigated.
2. Experimental section 2.1 Materials and Reagents. Compound 2',7'-dibromo-9,9'-spirobi[fluorene]-2,7-dicarbaldehyde (1) was prepared according to our previous method [2, 21]. All chemicals were commercially available and of analytical grade. Solutions 5
of metal ions were all prepared from their chloride salts. Ultrapure water was used from a Millipore water purification system.
2.2. Instruments and Measurements. Melting points were determined with an XT-4A apparatus and are uncorrected. 1H NMR and 13C NMR spectra were obtained on a Bruker DRX 400 MHz spectrometer. Chemical shifts were reported in ppm relative to a Me4Si standard. Steady-state emission spectra were recorded on Perkin Elmer LS55 instrument. Visible absorption spectra were determined on Perkin Elmer Lambda 35 spectrophotometer. Two-photon absorption cross-section was measured by the two-photon excited fluorescence (TPEF) technique using a Ti:Sapphire laser (Spectra-Physics). This laser provided pulses of 100 fs of duration with repetition rate of 80MHz at wavelength of 800 nm. The laser beam was focused into a quartz cell of 1cm path length by using a 5 cm focal-length lens. A half-wave plate and a polarizer were used to control the excitation intensity. The induced two- photon fluorescence was collimated by a lens at a direction perpendicular to the pump beam. To minimize the attenuation of fluorescence due to linear absorption effects, the excitation beam was focused as close as possible to the lateral wall of the quartz cell. The TPEF was then focused into the input slit of an imaging spectrograph and recorded at the exit with a CCD camera. To calculate the TPEF cross sections, Rhodamine B in methanol solution (10 mM) was utilized as references for the calculation. All the samples and standards were tested under the same experimental conditions. The TPEF cross-sections were calculated by the equation:σTPEF=σref cref nref F/cnFref, where c and n were the concentration and refractive index of the samples and reference, and F was the integral of the TPEF spectrum. The two photon cross-section σ was then calculated by the equation σ =σTPEF/Φ, where Φ was the fluorescence quantum yield.
6
2.3 Synthesis of SPF-TSA 2.3.1
Preparation of compound 2
To a solution of 1(52.8mg, 0.1mmol) and methyl thioglycolate (45μl, 1mmol) in dry dichloromethane (2 ml), BF3.Et2O (65μl, mmol) was added. After stirring at 0℃ for 2 h, the reaction was quenched by addition of saturated NaHCO3. The mixture was extracted with dichloromethane (10 ml×3). The combined organic phase was washed with brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, and purified by silica gel column chromatography using petroleum ether/ethyl acetate/dichloromethane (80:1:4-20:5:3,v/v) to yield 60 mg
of 2 as white solid (65.4%). Mp:143~145℃. 1H NMR (400 MHz,
CDCl3) δ 7.81 (d, J = 8.0 Hz, 1H ), 7.69 (d, J = 8.0 Hz,1H ), 7.56-7.48 (m, 2H ),6.80 (d, J = 1.6 Hz, 1H ), 6.72 (s, 2H ), 5.17 (s, 1H ), 3.60 (s, 6H ), 3.35 (d, J = 12.0 Hz, 2H ), 3.08 (d, J = 16.0 Hz, 2H ). 13C NMR (101 MHz, CDCl3) δ 170.38, 149.88, 148.15, 141.51, 139.93, 138.95, 131.65, 128.58, 127.42, 123.75, 122.23, 121.90, 121.01, 65.68, 53.71, 52.64, 33.86.
2.3.2
Preparation of compound 3
A mixture of compound 2 (300mg, 0.327mmol), 4-(diphenylamino)phenylboronic acid (207.8mg, 0.72 mmol), Pd(OAc)2 (4mg, 0.017 mmol), PPh3 (16.7mg, 0.068 mmol) and Na2CO3 (148mg,1.32mmol) in dry THF (20 ml) were reacted under nitrogen atmosphere. After the mixture was refluxed at 80℃ for 18 h, the mixture was cooled to room temperature. The solvent was evaporated and the residue was subjected to column chromatography on silica gel (petroleum ether: ethyl acetate:dichloromethane = 50:1:3-30:5:6, V/V) to afford yellowish solid (300mg, 73.7 %). Mp:80-82℃. 1H NMR (400 MHz, CDCl3) δ 7.90 (d, J = 8.0 Hz, 2H ), 7.81 (d, J = 8.0 Hz, 2H ), 7.60 (dd, J = 8.0, 1.6 Hz, 2H ), 7.51 (dd, J = 8.0, 1.6 Hz, 2H ), 7.28 (d, J = 8.0
7
Hz, 4H ), 7.22 (t, J = 8.0 Hz, 8H ), 7.10-7.05 (m, 8H ), 7.04-6.95 (m, 8H ), 6.85 (d, J = 1.2 Hz, 2H ), 6.79 (d, J = 1.2 Hz, 2H ), 5.14 (s, 2H ), 3.52 (s, 12H ), 3.32 (d, J = 16.0 Hz, 4H ), 3.05 (d, J = 16.0 Hz, 4H ).
2.3.3
Preparation of SPF-TSA
A mixture of 3 (26mg, 0.02 mmol) and aq. NaOH (0.15 ml, 1 N) in methanol (3 ml) was stirred for 10 h at rt. Then, pH of the solution was neutralized by aq. HCl (1 M). Na2CO3 was added to the solution to obtain sodium salt of SPF-TSA. Then the mixture extracted with ethylacetate (20 ml×3) for purification. The aqueous phase then was neutralized again with aq. HCl and extracted with ethylacetate. The extract was dried over Na2SO4. The solvent was removed at reduced pressure to afford SPF-TSA with 42% yield as a yellowish solid. Mp:172-173℃. 1H NMR (400 MHz, DMSO): δ 12.51 (s, 4H ), 8.11 (d, J = 8.0 Hz, 2H ), 8.03 (d, J = 8.0 Hz, 2H ), 7.70 (d, J = 8.0 Hz, 2H ), 7.46 (d, J = 8.0 Hz, 2H ), 7.37 (d, J = 8.0 Hz, 4H ), 7.27 (t, J = 8.0 Hz, 8H ), 7.05-6.95 (m, 12H ), 6.91 (m, J = 12.0 Hz, 4H ), 6.81 (s, 2H ), 6.70 (s, 2H ), 5.19 (s, 2H ), 3.25 (d, J = 16.0 Hz, 4H ), 3.12 (d, J = 16.0 Hz, 4H ). 13C NMR (101 MHz, CDCl3) δ 176.91, 150.09, 149.14, 147.79, 147.45, 141.96, 140.83, 140.61, 138.56, 135.07, 129.50, 128.44, 127.94, 127.14, 124.62, 124.00, 123.93, 123.20, 122.26, 120.93, 120.86, 66.37, 52.46, 33.50. Elemental analysis (%) calcd for C71H54N2O8S4: C 71.57, H 4.57, N 2.35, O 10.74, S 10.76. Found: C 91.00, H 6.37, N 3.05. TOF MS ( ES+ ): 1190. 3.
2.4. Confocal Imaging in Live Cells. 293T cells were grown in Dulbecco’s modified Eagle’s (DMEM) containing 4500 mg/L glucose, 3.7 g/L NaHCO3, 2 units/mlnystatin, 50 μg/ml gentamicin, 10 mM Hepes, and 10% fetal bovine serum at 37℃ in a humidified atmosphere containing 5% CO2. Two groups of cells were exposed to dye (50 μM) for 30 min and then washed three times with PBS buffer to remove excess probe, and one group of cells were incubated 8
with Hg2+ (100 μM) for 30 min and then washed with PBS buffer to remove unreacted Hg2+. TPEF images were taken on an Ultimal IV confocal microscope equipped with a femtosecond Ti:sapphire laser.
3. Results and discussion 3.1. Linear absorption and one-photon-excited fluorescence
The linear spectroscopic properties of SPF-TSA were measured in five organic solvents of different polarities with the concentration of 1.0× 10-5 mol L-1 (Table 1). It can be seen from Fig.1 and Table 1, SPF-TSA and SPF-TP have very similar absorption and emission spectral properties. The spectral features of
SPF-TSA were influenced only slightly by dithioacetal moiety.
The absorbance bands of
SPF-TSA
and SPF-TP located at the high-energy side with peaks around 300nm are mainly attributed to n-π* transitions derived from the triphenylamine unit [22], whereas the low-energy bands around 328-415 nm result from the π-π* transition of the triphenylamine-capped biphenyl branches [23]. The absorption spectra of SPF-TSA present a small shoulder peak at about 320 nm ( Fig.1), which can be attributed to the dithioacetal moiety [16, 24]. The PL emission spectra
of SPF-TSA
show significant red-shifts from
low-polarity to high-polarity solvents along with decreased PL intensity (Table 1), which indicates that the compound has a highly charged excited state due to photo induced charge transfer [25] .
3.2 Selective recognition of Hg2+
The sensing ability of SPF-TSA for metal cations (Li+, Na+, Co2+, Cu2+, Ca2+, Cd2+, Ag+, Zn2+, Hg2+, Pb2+, Mg2+, Mn2+, Ni2+, Fe3+) were investigated. When 2.0× 10-5M of Hg2+ ions were added to SPF-TSA in DMSO/water (1:1, V/V)
solution (1.0× 10-5M), the absorption and fluorescence spectra underwent
dramatic changes and the values of the molar absorptivity and the relative fluorescence intensity were decreased (Fig.2). The absorption bands at 370 nm decreased with simultaneous disappearance of the small 9
shoulder peaks at about 320 nm, generating an isosbestic point at 395 nm (Fig. 2a, Fig.S1). According to the literature [14,16,24],
SPF-TSA
underwent a mercury-promoted hydrolysis as shown in Scheme 3,
giving the product SPF-DA at room temperature. The sensing mechanism of SPF-TSA is confirmed by the NMR data of SPF-DA (Fig. S2 and Fig.S3), which were recorded after reaction. Further evidence for the formation of
the hydrolysis product
SPF-DA
was provided by the appearance of an isosbestic point in
the absorption spectra of SPF-TSA upon addition of Hg2+ (Fig. 2a, Fig.S1).
Along with the addition of
Hg2+, a color change from yellowish to colorless was observed (Fig.2a, inset), which suggested that SPF-TSA could serve as a “naked-eye” sensor for the detection of
Hg2+.
As depicted in Fig. 2a, Ag+,
Pb2+ and Ni2+ had a slight effect on the absorption of SPF-TSA. Upon addition of Hg2+, distinct fluorescent quenching can be observed
under the UV light (λ = 365 nm) (Fig.2b, inset). The fluorescence emission
change, formed upon addition of different concentration of Hg2+ to a solution of SPF-TSA (1.0× 10-5M), is shown in Figure 3. The fluorescence became weak gradually and blue-shifted with the increase of concentration. The fluorescence
the Hg2+
ratios (Io– I) / Io toward the surveyed metal ions are displayed in Fig.4.
The results show highly selective response of receptor SPF-TSA to Hg2+ as compared to the other metal ions −
−
(10-5M, 1:1, V/V) as a function of Hg2+ concentration in the range of 1 ×10 6 -1×10 5 M. [26]. A good linearity between Io / I and concentration of Hg2+ in the range of 1 ×10−6 -1×10−5 mol/L was obtained with a linearly dependent coefficient R2 of 0.992 (Fig. 5). Hence, a quantitative measurement of [Hg2+] was possible in DMSO/water (1:1, V/V)
in this concentration range [13]. The detection limit (DL)
was calculated by the following equation: DL = 3σ s/ms, where σs is the standard deviation of
blank sample
and ms is the slope of calibration curve. The detection limit of SPF-TSA was at least 2.6×10-8M, which is better than many reported data [26-30].
3.3. pH-dependent spectral properties For the analysis of pH-dependent properties, the spectra 10
of
SPF-TSA were recorded in DMSO-water
mixture (1:1, v/v, 1.0× 10-5M) between pH 1.01 and 13.68 using HCl and NaOH for pH adjustment (Fig. 6, Fig.7). In alkaline media (pH˃7.65), SPF-TSA showed no interesting responses toward pH variations. Therefore, the following studies were restricted to the survey of the spectral behavior of
SPF-TSA in
acidic condition. It can be seen from Fig. 6, with decreased pH, the absorption bands of SPF-TSA become weak and the low-energy band at 366 nm is red shifted gradually. When the pH value reached 1.01, the low-energy absorption band of SPF-TSA SPF-TSA
appeared at around 386 nm. In contrast, the fluorescence spectra of
are characterized by hyperchromic shifts with the decrease of the pH (Fig.7). When the pH is
decreased from 6.20 to 5.40, the fluorescence emission intensities of SPF-TSA decreased significantly. An anaysis of
F470 versus pH was shown in Fig.7 (inset) and the pKa value of
SPF-TSA determined by
fluorescence titration plots was found to be comparable at ∼6.00 [27,31,32]. This result
indicates that
SPF-TSA is suitable to detect acidic regions in cells and tissues. The pH-dependent spectral properties of SPF-TSA may be due to the protonation of
the nitrogen atom of
triphenylamine moiety under acidic
condition. It has been pointed out that the sensing procedure of Hg2+ is usually affected by solution pH value [33-35]. The emission intensities of
SPF-TSA with and without Hg2+ in phosphate buffered saline under various
pH values are shown in Fig. 8. There was no dramatic spectral change for SPF-TSA at higher than pH 8. From the view of sensitivity, the suitable range of pH for Hg2+ determination is found to be within 8-11 [35-38]. The sensing operations can be carried out in buffer solutions so that the influence from various pH values can be avoided [33].
3.4.Two-photon absorption cross-section and two-photon fluorescence
11
cell imaging
The two-photon cross-section values were measured by two-photon excited fluorescence (TPEF) technique in THF at a concentration of 1.0 ×10-5mol L-1 [31]. The values are 248GM (1 GM= 1×10-50cm4 s per photon) for SPF-TSA and 686GM for SPF-DA at 800 nm, respectively. SPF-TSA emits blue up-converted fluorescence with the maximal peak at about 435 nm and the profile is similar to that of the single-photon induced fluorescent spectrum (Fig.1, Fig.9a). However, the two-photon induced emission spectrum of SPF-DA in THF is different from that data obtained by one-photon excitation in the same solvent (Fig.9a, Fig.S4). Two emission bands at 429nm and 556nm were observed for SPF-DA on one-photon excitation
in THF (Fig.S4). It can be seen from Fig. 9b, SPF-DA exhibited only one emission
band located at ca. 575nm on 800nm laser excitation [32].
The result could be explained as follows (i) The
two-photon induced emission band located at 575nm may be the ICT (intramolecular charge-transfer) emission band. This viewpoint can be supported by the molecular structure and one-photon spectra of
fluorescence
SPF-DA. From the point of view of molecular structure, SPF-DA is a spiro-configured donor
(D)-acceptor (A) compound, in which electron-rich triphenylamine
and electro-deficient formyl moieties
are present on the two respective biphenyl branches of a 9,9'-spirobifluorene core [39]. The strong electron-withdrawing effect of the formyl group is beneficial for promoting interaction between the donor and acceptor parts in the excited state [32, 40]. Such spirobifluorene-bridged bipolar systems usually exhibit an efficient photoinduced excitation characteristic of a ICT emission band [32, 40]. As depicted in Fig.S4, the emission band at ca.525nm on single-photon excitation showed significant red-shifts from low-polarity to high-polarity solvents along with decreased PL intensity, supporting the assignment of
it to ICT
process [25]. The photo induced charge transfer would be happened in the same way for two- photon excitation and the ICT band (at 575nm) increases significantly due to the intense laser, while the normal emission band maximized at about 450nm was too weak to be 12
observed (Fig.9b) [32]. Or (ii) The large red
shift of the two-photon emission of SPF-DA may be due to some structural change of the molecule by high laser intensity in the two- photon absorption event. The two-photon showed about 140nm red-shifted as compared to that
induced emission of
SPF-DA
of SPF-TSA. The emission wavelength change
(140-nm shift) before and after addition of Hg2+ is larger than the reported two-photon fluorescent probes [16]. The TPEF intensity of SPF-TSA was decreased upon the addition of mercury ion and acidification,
respectively (Fig.10 and Fig.11). The results suggest that SPF-TSA can be used as an efficient two-photon fluorescence probe for the detection of Hg2+ and proton [41].
The pH values of most biological samples are
within the range 5.25-8.93 [36]. The sensitivity of
SPF-TSA to pH variations is detrimental to the detection of Hg2+ in living cells. Fortunately, SPF-TSA is
a reaction-based two-photon probe
for mercury species. Sensing of Hg2+ ions in cells by two-photon
microscopy can be achieved via a emission change with a significant wavelength shift. Thus, we have demonstrated the feasibility of the application of SPF-TSA to the two-photon fluorescence imaging of Hg2+ present in live
cells [14, 42]. Two groups of 293T cells were exposed to dye (50μM) for 30 min and
then washed three times with PBS buffer to remove excess dye, and one group of cells were incubated with Hg2+ (100μM) for 30 min and then washed with PBS buffer to remove unreacted Hg2+. The cells treated with the dye alone show bright blue fluorescence by two-photon microscopy (Fig.12, left). The cells treated with Hg2+ show yellow-green fluorescence owing to the hydrolysis product SPF-DA (Fig. 12, right). The results demonstrate that SPF-TSA enables fluorescence imaging of both the probe and Hg2+ in cells. The cytotoxic effect of SPF-TSA against 293T cells was assessed by MTT assay over a 24 h period. 293T cells incubated with 2.5 mM of SPF-TSA remained 90% viable after 24 h of feeding time (In contrast, 100% survival is observed for 293T cells not treated with SPF-TSA). The result suggests that SPF-TSA exhibits low toxicity 13
to 293T cells in our measuring range and could be used to detect Hg2+ in vivo with little damage [13,43].
4. Conclusions
We have developed a spirobifluorene-based fluorescence probe. In comparison to the reported results, SPF-TSA is distinguished by exclusive selectivity and sensitivity for mercury species, large two- photon absorption cross-section , and large emission wavelength change (140-nm shift) before and after addition of Hg2+ . SPF-TSA enable fluorescent imaging of both the probe and mercury ions in cells by two-photon microscopy . In addition,
SPF-TSA posseses excellent indicator properties within the acidic
pH-range. The results demonstrated that spirobifluorene is a suitable fluorophore core for highly sensitive two-photon fluorescent probes.
Acknowledgments
This work was financially supported by the Natural Science Foundation of Shanghai City of China (No.15ZR1431400).
Appendix A. Supplementary data Supplementary data related to this article can be found at
14
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Biomol.Chem. 12(2014) 4550-4566. [43] Z. P. Yu, Z. Zheng, M. D. Yang, L.K. Wang, Y. P. Tian, J. Y. Wu, H.P. Zhou, H.M. Xu, Z.Q. Wu, Photon-induced intramolecular charge transfer with the influence of D/A group and mode: optical physical properties and bioimaging. J. Mater. Chem. C. 1(2013) 7026-7033.
17
Biographies Haibo Xiao received his PhD in chemistry from East China University of Science and Technology in 2005. He is currently an associate professor of the chemistry department at Shanghai Normal University. His main research fields are chemical sensors, organic nonlinear optical materials and organic light-emiting material. Yanzhen Zhang is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Shaozhi Li is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Wu Zhang is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Zhongying Han is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Jingjing Tan is a Master course student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Shenyao Zhang is a student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors. Jingyan Du is a student at Shanghai Normal University. His research interest is to develop highly selective fluorescence sensors.
18
SPF-TSA SPF-TP
1.0
0.8
0.8
0.6
0.6
0.4
0.4
0.2
0.2
0.0 300
350
400 450 Wavelength (nm)
500
550
Emission Intensity (arb.unit.)
Absorbance
1.0
0.0
Fig. 1. Normalized one-photon absorption and fluorescent emission of SPF-TSA and SPF-TP in THF.
19
0.8 Hg
0.7
Absorbance
0.6 0.5
blank + Ag 2+ Ca 2+ Cd 2+ Co 2+ Hg + Li 2+ Mg 2+ Mn + Na 2+ Ni 2+ Pb 2+ Zn 3+ Fe 2+ Cu
2+
a)
0.4 0.3 0.2 0.1 0.0 325
350
375 400 Wavelength(nm)
425
450
Fig.2 (a)The absorption spectra of SPF-TSA upon addition of various metal ions in DMSO/water (1:1, V/V) solution (1.0× 10-5M). Inset: the photograph
of SPF-TSA solution (1.0× 10-5M) in the presence of
2.0× 10-5M of Hg2+. (b)The fluorescence spectra of SPF-TSA upon addition of various metal ions in DMSO/water (1:1, V/V) solution (1.0× 10-5M). -5
-5
10 M) in the presence of 2.0× 10 M
20
2+
Inset: the photograph
Hg under the UV light.
of SPF-TSA solution
(1.0×
blank 1×10-7 5×10-7 1×10-6 2×10-6 4×10-6 6×10-6 8×10-6 1×10-5 2×10-5 4×10-5 6×10-5 8×10-5 1×10-4 2×10-4
Fluorescence Intensity
1600
1200
800
400
0 400
450
500
550
600
Wavelength(nm)
Fig.3. Fluorescence emission spectra of SPF-TSA(1.0× 10-5M) upon addition of
21
Hg2+ in
various concentrations.
0.8
(I0-I)/I0
0.6
0.4
0.2
0.0
Blank Ag[I] Ca[II]Cd[II]Co[II]Hg[II] Li[I] Mg[II]Mn[II] Na[I] Ni[II] Pb[II] Zn[II]Fe[III]Cu[II]
Fig.4. Fluorescence ratio (Io– I) / Io of receptor SPF-TSA upon addition of a particular metal salt in DMSO/water (1:1, V/V, 1.0× 10-5M ).
22
20 5
2+
18
Y = 1.598x10 [Hg ]+ 3.10459
16
R =0.9922
2
I0/I
14 12 10 8 6 4 0
200
400
600 2+
800
1000
-8
[Hg ]/10 M
Fig.5. The relative fluorescence intensity (I0/I) of SPF-TSA
23
DMSO/water solution
0.55
pH=7.65
0.50 0.45
Absorbance
0.40 0.35 0.30 0.25
pH=1.0
0.20
1.01 1.50 2.02 3.01 3.98 5.00 6.20 6.96 7.65
0.15 0.10 0.05 0.00 300
350 400 450 Wavelength(nm)
500
550
Fig. 6. Absorption spectra of SPF-TSA (1.0 ×10-5M) under acidic conditions.
24
2000
2000 1800
pH=7.65
1600
Intensity
1400 1200 1000
1.01 1.50 2.02 2.55 3.01 3.98 4.20 5.00 5.40 6.20 6.96 7.65
Fluorescence Intensity
800
1500
600 400 200 2
4
6
8
10
12
14
pH
1000
500
pH=1.01
0 400
450
500
550
600
Wavelength(nm)
Fig. 7. One-photon-excited fluorescence spectra of SPF-TSA (1.0 ×10-5M)
25
under acidic conditions..
0
2
4
6
8
10 10
2000 SPF-TSA SPF-TSA+Hg
Emission Intensity (a.u.)
1800
8
1600 1400
6
1200 1000
4
800 600
2
400 200 0
0
1
2
3
4
5
6
7
8
9
10
11
12
pH
Fig. 8. Luminescence intensity of SPF-TSA (1.0 ×10-5M) at 470 nm in the absence and presence of 5 equiv of Hg2+ as a function of pH. Conditions: λex 360 nm.
26
3500 2
4
6
8
10
0
10
4
3.6 2
3.4 3.2 1.6
6000 4000
0
1.7
1.8
1.9
2.0
2.1
2.2
logI 0
300mw 250mw 200mw 150mw 100mw 50mw
a)
2000
400
450
500
Wavelength(nm)
550
600
2500
6
8
10 10
200mw 250mw 340mw 410mw 480mw
8
2.78
3000
Fluorescence Intensity
3.8
4
Experimetal data Slope=0.20
8
6
8000
2
2.80
Experimental data Slope=1.89
4.0
logIF
Fluorescence Intensity
0
4.2
6
logIF
4.4
10000
2.76 4
2.74
2
2.72 2.70 2.0
0
2.1
2.2
2.3
2.4
2.5
logI0
2000 1500
b)
1000 400
450
500
550
600
650
700
750
Wavelenth(nm)
Fig.9.Two-photon excited fluorescence spectra of SPF-TSA (a) and SPF-DA (b) under different pumped powers at 800 nm. Inset, power dependence ofthe TPA-induced up conversion emission intensity on the input intensity.
27
Fluorescence intensity（ Counts）
0
2
4
6
8
blank 4.0×10-6 8.0×10-6 1.5×10-5 2.0×10-5 2.2×10-5 2.4×10-5 2.6×10-5 2.8×10-5
5000 4000 3000 2000
10 10
8
6
4
2
1000 0
0
400
450
500
550
600
Wavelength(nm)
Fig.10. The two-photon fluorescence spectra of SPF-TSA (1.0 ×10-5M ) in DMSO/water (1:1,v/v) upon the addition of mercury ions (λex= 800 nm).
28
0
2
4
6
8
10 10
5000
Fluorescence Intensity
pH=7.03
7.03 6.90 6.25 5.41 4.61 3.72 2.87 2.01
4000
3000
2000 pH=2.01
1000
0
8
6
4
2
0
450
500
550
600
Wavelenth (nm)
Fig.11. The two-photon fluorescence spectra of SPF-TSA (1.0 ×10-5M ) in DMSO/water (1:1,v/v) under acidic conditions (λex= 800 nm).
29
Fig. 12.Two-photon
microscopy images of 293T cells labeled with SPF-TSA before (left)
and after (right) addition of
30
Hg2+ (100 μM). Excitation at 800 nm.
Scheme 1. The structures of SPF-TP.
31
Scheme 2. The synthesis route of
32
SPF-TSA.
Scheme 3. Mercury ion-promoted hydrolysis of
33
SPF-TSA.
Table 1. Linear Optical Properties of solvent
λabs (nm)
λfl(nm) a
Φb
Toluene
296, 370
426
0.88
THF
296, 368
428
0.85
CH2Cl2
295, 372
441
0.81
DMF
296, 374
454
0.70
Toluene
301, 372
418
0.98
THF
297, 369
425
0.90
CH2Cl2
296, 369
438
0.87
DMF
298, 373
458
0.70
compound
SPF-TSA
SPF-TP
a
excited at 370 nm.
b
φ Fluorescence quantum yield, was dtermined by using Rhodamine B as the standard.
34
SPF-TSA and SPF-TP.