Design of Ratiometric Emission Probe with Visible Light Excitation for

Jul 23, 2014 - Up to now, the most successful approach for [Ca2+]i measurement is to introduce optically sensitive Ca2+ probes into living cells and t...
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Design of Ratiometric Emission Probe with Visible Light Excitation for Determination of Ca2+ in Living Cells Qiaoling Liu,†,‡ Huizhi Du,† Xiaoze Ren,§ Wei Bian,†,§ Li Fan,† Shaomin Shuang,† Chuan Dong,*,† Qin Hu,¶ and Martin M. F. Choi*,¶ †

Institute of Environmental Science, and School of Chemistry and Chemical Engineering, Shanxi University, Taiyuan 030006, China Department of Chemistry, Taiyuan Normal University, Taiyuan 030031, China § School of Basic Medical Science, Shanxi Medical University, Taiyuan 030001, China ¶ Partner State Key Laboratory of Environmental and Biological Analysis, and Department of Chemistry, Hong Kong Baptist University, 224 Waterloo Road, Kowloon Tong, Hong Kong SAR China ‡

S Supporting Information *

ABSTRACT: An organic salt as a fluorescent probe based on intramolecular charge transfer for Ca2+ determination is developed. Ca2+ can be detected by ratiometric emission at 490 and 594 nm with an excitation wavelength of 405 nm. This probe is highly selective for Ca2+ over other divalent metal cations and displays a large Stokes shift of 189 nm that can avoid interference of the excitation light beam and autofluorescence of biological samples. The dissociation constant for Ca2+ is 2.25 ± 0.47 μM and pertinent to Ca2+ detection in cellular resting and dynamic states. The probe demonstrates its application in monitoring Ca2+ in living cells under confocal microscopic imaging.

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(BAPTA) has been synthesized successfully as a Ca 2+ ionophore,8 while fluorescein, rhodamine, quinine, indole, and salicylaldehyde derivatives have been selected as the fluorophores to fabricate Ca2+ probes, namely fluo-3, fluo-4,9 rhod-1, rhod-5N,10−12 quin-2,13,14 indo-1, indo-5F,15,16 and fura-2,17 respectively. The detection approach for these Ca2+ probes is either based on intensity or ratiometric measurements. It is generally accepted that the latter is much better since the probes not only exhibit large spectral shifts but also provide ratiometric intensity measurements at two excitation or emission wavelengths instead of the absolute intensity of one band; thus, internal calibration of Ca2+ can be in situ realized. Ratiometric measurement can cancel out most or all possible variations in instrument performance, sample thickness, and probe concentration.18,19 Among these Ca2+ probes, ratiometric emission is better than ratiometric excitation, since it is more convenient to monitor dual emission light under one single excitation light. To our knowledge, only Indo-1 and Indo-5F can perform ratiometric emission measurements. Unfortunately, both probes require UV excitation and their dissociation constants (Kd) are relatively small (0.23 and 0.47 μM, respectively). UV excitation often results in strong autofluorescence interference with biological samples. Small Kd means the probe could saturate quickly if the Ca2+ concentration is too

alcium ion (Ca2+) as a versatile intracellular signal messenger plays an indispensable role in the human body. It participates in numerous cellular processes, including fertilization, development, differentiation, and cell death by changing Ca2+ cytoplasmic concentrations to control different cellular functions.1−3 The total calcium content in resting cells is typically 1−7 mM. Most of them (>99.9%) are stored in organelles or bound to membrane components, cytosolic metabolites, and proteins. The internal free Ca2+ is only around 0.10 μM. During various cellular activities, however, the free Ca2+ concentration could be regulated to 10−100 folds by the release of Ca2+ from the intracellular Ca2+-storing organelles or by the influx of extracellular calcium.4,5 Since the changes in cytosolic Ca2+ concentration are transitory, tracking the simultaneous changes of intracellular Ca2+ concentration [Ca2+]i and understanding the signal Ca2+ pathways is of particular importance. This will certainly help us understand the Ca2+ functions and mechanisms at the cellular level. Up to now, the most successful approach for [Ca2+]i measurement is to introduce optically sensitive Ca2+ probes into living cells and then monitor the fluorescence changes of the probes by digital imaging microscopy. Fluorescent probes based on metal-induced fluorescence change are attractive, owing to their simple operation, low detection limit for metal ions, and capability of real-time detection.6,7 To design an effective fluorescent probe, it is essential to have an ion recognition unit (ionophore) and a fluorophore. So far, 1,2bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid © 2014 American Chemical Society

Received: January 27, 2014 Accepted: July 23, 2014 Published: July 23, 2014 8025

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high and then is unable to monitor fast Ca2 transient, resulting in inaccurate measurement. 1,3,4-Oxadiazole derivatives (OXO) have been recently used as electroluminescent material attributing to the high electron affinity of the oxadiazole ring, excellent thermal stability, and favorable optical properties.20,21 In addition, OXO shows some promising biological functions of antiinflammatory, antimicrobial, antiviral, anticonvulsant, and anticancer properties.22,23 In this work, we propose to incorporate two OXO as the bifluorophore with BAPTA as the ionophore to design a new Ca2+ probe. It is anticipated that the proposed Ca2+ probe will display low toxicity and possess favorable optical properties for [Ca2+]i detection. Herein, two 2-(4-ethoxyphenyl)-5-(4-methylphenyl)-1,3,4-oxadiazole units are incorporated with a BAPTA moiety to form a new sandwich molecular structure, namely OBO. This novel Ca2+ probe displays a large Stokes shift after binding with Ca2+. Scheme 1 illustrates the chemical structure and reaction of OBO with Ca2+. When OBO binds with Ca2+, the electron-

Supporting Information. Deionized water was obtained from a Milli-Q water purification system (Millipore, Bedford, MA). 50 mM 2-[4-(2-Hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer solution (pH 7.2) containing 0.10 M KCl and 10 mM ethylene glycol-bis(2-aminoethyl ether)N,N,N′,N′-tetraacetic acid (EGTA) was used to prepare the OBO and Ca2+ standard solutions. The standard solutions of 0.0−11.1 μM Ca2+ were obtained by serial dilutions of 0.20 M Ca2+ solution in a 50 mM HEPES buffer solution containing 0.10 M KCl and 10 mM EGTA at pH 7.2.8,15 Stock solutions (0.20 M) of Ca2+, Mg2+, Zn2+, Co2+, Mn2+, Ba2+, Ni2+, Sr2+, Cu2+, Hg2+, and Pb2+ were prepared from their chloride salts. Freshly prepared just before the experiment were 0.20 M Fe2+ and 1500 units/mL penicillin G sodium salt solutions. Instrumentation. UV−vis absorption spectra were recorded on a Puxi TU-1901 UV−vis absorption spectrophotometer (Beijing, China). Steady-state fluorescence measurements were performed on an Edinburgh FLSP 920 spectrofluorometer (Livingston, U.K.) at 21 ± 1 °C. 1H and 13 C NMR spectra were recorded on a Bruker AVANCE III 600 NMR spectrometer (Rheinstetten, Germany). Elemental analysis was conducted on an Elementar Vario EL Cube CHNOS analyzer (Hanau, Germany). Mass spectrum was acquired on a Bruker Autoflex matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer (Bremen, Germany). Cell Cytotoxicity Assay. The MTT assay [3-(4,5dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide) was used to assess the cytotoxicity of OBO to human umbilical vein endothelial cells (HUVEC). HUVEC (8 × 104 cells/well/ 200 μL) were cultured in a 96-well plate at 37 °C in a 5.0% CO2 atmosphere overnight and then exposed to various concentrations of OBO (0.010, 0.10, 1.0, and 10.0 μM) for 48 h. Cells treated with medium only served as a negative control group. After washing the cells with the HEPES buffer solution (pH 7.2) three times, 10 μL of MTT solution (10 mg/ mL) was introduced into each well of the 96-well microplate for another 5 h. Then the remaining MTT solution was removed from the wells, and 150 μL of DMSO was added into each well to dissolve the intracellular blue-violet formazan crystals, and the absorbance was measured by a microplate reader at a wavelength of 490 nm. The cell viability was determined as a percentage of the untreated control cells by dividing the mean absorbance of each treatment by the mean absorbance of the untreated cells. All treatments were tested with three independent experiments. Cell Culture and Imaging. HUVEC were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal bovine serum (Life Technologies, Grand Island, NY). One day before cell imaging, HUVEC were seeded on coverslips. The next day, HUVEC were incubated with 5.0 μM OBO− ester for 35 min at 37 °C under 5.0% CO2, washed with HEPES buffer three times, and then subjected to cell imaging under an Olympus FluoView 1000 CLSM (Center Valley, PA) using an objective lens (20× ) and an excitation light beam of 405 nm, and the emissions were centered at 480−510 and 580−610 nm. Then 30 units/mL penicillin G sodium salt was added to these OBO−ester incubated cells. The image and emission intensities were simultaneously recorded to monitor the transient changes in [Ca2+]i for 180 s.

Scheme 1. Schematic Illustration for the Reaction of OBO and Ca2+a

a The images display the fluorescence of OBO (left) and the OBO− Ca2+ complex (right) under UV lamp irradiation.

donating ability of BAPTA reduces with a concomitant effect on the hypsochromic shift of the absorption and emission spectra of OBO; as such, it can function as a sensitive and selective Ca2+ probe. The major attribute is that OBO and the OBO−Ca2+ complex possess two different strong emission bands in the visible region under one single visible light excitation. This means that ratiometric emission measurement of Ca2+ is possible with negligible background fluorescence interference. The developed Ca2+ probe has been successfully applied to monitor the transient changes of [Ca2+]i in living cells by a confocal laser scanning microscope (CLSM). The most important merits of this probe are its good watersolubility, nontoxicity, strong emissions, and visible light excitation.

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EXPERIMENTAL SECTION Chemicals and Materials. All chemicals were purchased from Shanghai Aladdin Reagent Co., Ltd. Solvents (AR grade) were purchased from commercial suppliers without further purification before use. The synthetic procedures of OBO− ester (the OBO precursor) and OBO are deposited in the 8026

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Technical Note

RESULTS AND DISCUSSION The optical properties of OBO are first examined. The absorption spectra of OBO in the HEPES buffer solution (50 mM, pH 7.2) with various concentrations of Ca2+ are displayed in Figure 1. Free OBO shows two strong absorption bands

Figure 2. Fluorescence emission spectra of 1.0 μM OBO in 50 mM HEPES (pH 7.2) containing 0.10 M KCl and 10 mM EGTA in the presence of various concentrations of Ca2+ (1−12:0.00, 0.038, 0.098, 0.15, 0.227, 0.341, 0.582, 0.906, 2.04, 3.56, 5.45, and 11.1 μM) at an excitation wavelength of 372 nm.

at 560 and 594 nm, respectively. F560/F594 increases with the increase in the concentration of Ca2+, inferring that OBO can function as an ideal probe for the ratiometric emission measurement of Ca2+. Similar observations are found at emission wavelengths of 480−554 and 594 nm (i.e., F480−554/ F594). Similarly, Figure 3 depicts the emission spectra of OBO in the physiological conditions at various [Ca2+]o. A strong

Figure 1. Normalized absorption spectra of 1.0 μM OBO in 50 mM HEPES (pH 7.2) containing 0.10 M KCl and 10 mM EGTA in the presence of various concentrations of Ca2+ (1−10:0.00, 0.098, 0.227, 0.341, 0.582, 0.906, 2.04, 3.56, 5.45, and 11.1 μM).

centered at 304 nm (ε = 3.82 × 104 M−1 cm−1) and 380 nm (ε = 5.82 × 104 M−1 cm−1), assigned to the π−π* transition and intramolecular charge transfer (ICT) band, respectively. Upon addition of Ca2+ (0.0−11.1 μM), the bands at 304 and 380 nm decrease gradually and the ICT band exhibits a hypsochromic shift to 352 nm (ε = 7.15 × 104 M−1 cm−1). When OBO binds with Ca2+, the electron-donating ability of the nitrogen atoms in the BAPTA moiety diminishes with a concomitant effect on widening the energy gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital of the ICT band. Two isosbestic points at 310 and 372 nm are observed, implying the conversion of orange-red OBO to a faint yellow OBO−Ca2+ complex. Figure S1 of the Supporting Information displays the plot of the ratiometric absorbance (A352/A405) at 352 and 405 nm, respectively, as a function of the concentration of Ca2+ ([Ca2+]o). At [Ca2+]o = 0.0−2.0 μM, A352/A405 increases rapidly with the increase in [Ca2+]o and then increases slowly at [Ca2+]o > 2.0 μM until it approaches the plateau at about 11 μM [Ca2+]o. These results indicate the formation of the OBO− Ca2+ complex from OBO and Ca2+. Similar results are found for monitoring the ratiometric absorbance (A352/A380) at 352 and 380 nm, respectively, as a function of [Ca2+]o. Herein, 405 nm is chosen in order to align with the use of laser-induced excitation to capture the living cells image incorporating with OBO−ester and Ca2+ under a CLSM (vide infra). Figure 2 depicts the emission spectra of OBO with various concentrations of Ca2+ under the isosbestic point excitation wavelength of 372 nm. A strong emission band with an emission maximum (λflmax) of 594 nm is observed, corresponding to the free OBO. On gradual addition of Ca2+ to OBO, λflmax is hypsochromically shifted and finally reaches 560 nm corresponding to the emission band of the OBO−Ca2+ complex. Figure S2 of the Supporting Information displays the plot of the ratiometric emission (F560/F594) against concentration of Ca2+, where F560 and F594 are the emissions

Figure 3. Fluorescence emission spectra of 1.0 μM OBO in 50 mM HEPES (pH 7.2) containing 0.10 M KCl and 10 mM EGTA in the presence of various concentrations of Ca2+ (1−12:0.00, 0.038, 0.098, 0.15, 0.227, 0.341, 0.582, 0.906, 2.04, 3.56, 5.45, and 11.1 μM) at an excitation wavelength of 380 nm.

emission band with λflmax of 594 nm corresponding to the free OBO is observed at the absorption maximum (380 nm) of OBO. On gradual addition of Ca2+ to OBO, λflmax is hypsochromically shifted and finally reaches 560 nm accompanying with the emergence of an isoemissive point at 554 nm. Figure S3 of the Supporting Information displays the plot of the ratiometric emission (F490/F594 ) at 490 and 594 nm, respectively, under 380 nm excitation as a function of [Ca2+]o. F490/F594 increases with the increase in [Ca2+]o, demonstrating again that OBO is an ideal probe for ratiometric emission measurement of Ca2+. Similar observations are found at F480−554/F594; however, F490/F594 produces the best response to Ca2+. As such, it was chosen for subsequent work. 8027

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light region, possesses a large Stokes shift, and a large Kd value, facilitating its uses in intracellular Ca2+ determination. The fluorescence quantum yields (Φ) of OBO and the OBO−Ca2+ complex are determined as 0.024 and 0.019, respectively. Quinine bisulfate (Φ = 0.546) is used as the fluorescence standard (eq 9 of the Supporting Information).24,25 The Φ of the OBO−Ca2+ complex is slightly lower than that of OBO, indicating that Ca2+ would not affect too much on the Φ of OBO. However, the Φ of OBO and the OBO−Ca2+ complex are smaller than that of other Ca2+ probes such as Indo-1 (0.38) and the Indo-1-Ca2+ complex (0.56). The smaller Φ might be attributed to the polar solvent−solute interactions with a concomitant effect on a reduced coplanarity, more efficient nonradiative deactivation, and finally a drastically reduced brightness.26 Fortunately, OBO can function well in visible light excitation, whereas the Indo dyes can only work in the UV region. The fluorescence lifetimes (τ) of OBO and the OBO−Ca2+ complex are determined as 1.41 and 1.42 ns, respectively, from time-resolved fluorescence spectroscopy. Not much difference between the τ of OBO and the OBO−Ca2+ complex is observed. The influence of pH on the probe OBO was investigated in the biologically relevant pH range (5.5−9.0) (Figure S9 of the Supporting Information). OBO shows the strongest absorptions at pH 7.0−7.5, which means that it is suitable for use in the physiological environment. Figure S10 of the Supporting Information summarizes the effect of various bivalent metal ions on F490/F594 of OBO. Among these cations, Ca2+ exhibits the largest response to OBO while Mg2+, Zn2+, Co2+, Mn2+, Fe2+, Cu2+, Ba2+, Ni2+, Sr2+, Hg2+, and Pb2+ have only slight effects. Moreover, when both Ca2+ and the bivalent metal ions coexist in HEPES solutions, OBO maintains larger responses to Ca2+, indicating that OBO is more selective to Ca2+ than other coexisted bivalent cations in physiological conditions. Since OBO shows high selectivity to Ca2+, it is possible to explore its use in intracellular Ca2+ imaging. Before that, it is crucial to assess the toxicity of OBO to HUVEC by MTT assay.27 Figure S11 of the Supporting Information depicts the cell viability studies under various concentrations of OBO. The cell viability maintains almost 100% as compared to the control at the concentrations of 0.010−10.0 μM OBO, indicating OBO does not display any toxicity to the HUVEC and inferring their potential use in intracellular imaging of living cells. To explore the potential application in living cells, OBO− ester is employed for cell permeability. HUVEC are incubated with OBO−ester (5.0 μM) at 37 °C for 35 min. Then the ester is converted to OBO inside the cells by intracellular esterase, ensuring that the Ca2+ signal could be deciphered in the cytosol. The confocal fluorescent imaging is captured by a 405 nm laser. The emission wavelengths are centered at 580−610 and 480−510 nm for studying the fluorescent properties of intracellular OBO and the OBO−Ca2+ complex. Figure 4 (panels a and b) depicts the images of the stained cells at resting state under the emission wavelengths of 580−610 and 480−510 nm, respectively. Figure 4a shows strong red emissions (free OBO) for the cells, whereas Figure 4b has very weak green emissions, indicating that the initial [Ca2+]i is low in the cytosol. Then penicillin G sodium salt (30 units/ mL) is administered to these stained cells and Figure 4 (panels c and d) depicts the images after 180 s. The red emission fades while the green emission (OBO−Ca2+ complex) glows, inferring the release of [Ca2+]i from the intracellular Ca2+storing organelles triggered by the penicillin G sodium salt.

Figure S4a of the Supporting Information depicts the emission spectra of OBO with various concentrations of Ca2+ at 405 nm excitation. The typical emission band of OBO at 594 nm is observed. On gradual addition of Ca2+ to OBO, the emission band decreases and shifts to a broad band at ca. 518 nm. Figure S4b of the Supporting Information displays the plot of the ratiometric emission (F490/F594) at 490 and 594 nm, respectively, under 405 nm excitation as a function of [Ca2+]o. F490/F594 increases with the increase in [Ca2+]o, demonstrating that OBO can function as a Ca2+ probe for ratiometric emission measurement of Ca2+ under visible light (405 nm) excitation. Herein, 405 nm is preferred, as it can be used in line with the CLSM for [Ca2+]i detection (vide infra). All the above spectrofluorometric data provide us with valuable information for exploring the potential use of OBO for Ca2+ detection. The developed Ca2+ probe is immune to interferences from the excitation light beam and autofluorescence background of the biological sample. A large spectral shift of 104 nm at 380 or 405 nm excitation between the two emission wavelengths (490/594 nm) results in large F490/F594 values. The stoichiometric ratio for the formation of the OBO−Ca2+ complex is determined as 1:1 by the Job’s method (Figure S5 of the Supporting Information). Its Kd is 2.25 ± 0.47 μM (eq 3 and Figure S6 of the Supporting Information), which is much larger than that of Indo-1 and Indo-5F (Table 1). As such, the Table 1. Working Wavelengths and Dissociation Constants of OBO and Indo-1/5F Ca2+ probe

excitation (nm)

emission (nm)

Indo-1 Indo-5F OBO

349 344 405

410/485 398/471 490/594

Stokes shift (nm) Kd (μM) 136 127 189

0.23 0.47 2.25

working [Ca2+]o range for OBO would be 0.225−22.5 μM as the [Ca2+]o should be 0.10−10 times the Kd. This working range is suitable for monitoring [Ca2+]i by OBO at cellular resting or dynamic state and this will be discussed in the later section. Since Mg2+ is well-known for causing interference on Ca2+ detection, the spectral response of OBO to Mg2+ is investigated (Figure S7a of the Supporting Information). The Hill’s plot determines that OBO forms a 1:1 complex with Mg2+. The Kd is 5.92 ± 0.345 mM (eq 3 and Figure S7b of the Supporting Information), which is much larger than that of the OBO−Ca2+ complex, implying that the binding of OBO to Mg2+ is much weaker than that of Ca2+ and thus will not pose any interference on Ca2+ detection. Figure S8 (panels a and b) of the Supporting Information depicts the emission spectra of OBO with various concentrations of Ca2+ and intracellular concentrations of Mg2+ (0.80 mM), K+ (139 mM), and Na+ (12.0 mM) at excitations 380 and 405 nm, respectively. The response of OBO to Ca2+ remains more or less the same, indicating that the intracellular Mg2+ and Na+ would not pose any interference on Ca2+ detection under an intercellular environment. The Kd is determined as 2.34 ± 0.44 μM by eq 3 and Figure S8c of the Supporting Information, which is very close to that without Mg2+ and Na+. Table 1 summarizes the working wavelengths and dissociation constants of Ca probes OBO and Indo-1/5F. The major advantages of OBO over other Ca2+ probes such as Indo-1 and Indo-5F are that OBO can function in the visible 8028

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Moreover, the real-time track of the [Ca2+]i signal in living HUVEC is another attractive attribute that can be further expounded. It is anticipated that OBO has great potential for applications in the biomedical and biological fields.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax: +86-351-7018613. *E-mail: [email protected]. Fax: +852-34117348. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported by grants from the Hundred Talent Programme of Shanxi Province and the National Natural Science Foundation of China (Grants 21175086 and 21175087).

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REFERENCES

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Figure 4. Confocal fluorescence images of intracellular Ca2+ in living HUVEC. (a and b) are HUVEC incubated with OBO-ester for 35 min at 37 °C and observed emission wavelengths of 580−610 nm and 480−510 nm, respectively. (c and d) are the same images captured after addition of penicillin G sodium salt (30 U/mL) for 180 s. (e) Fluorescence intensities (F490 and F594) are recorded at the marked circle areas 1 and 2 from 0 to 180 s at emission wavelengths of 594 and 490 nm, respectively. (f) Ratiometric fluorescence F490/F594 measurements at areas 1 and 2 from 0 to 180 s. The excitation wavelength is 405 nm.

These features dovetail nicely with the behavior of cytosolic free [Ca2+]i in HUVEC that ranges 59−80 and 675−1823 nM at resting and dynamic states, respectively.28 Figure 4e shows the emission intensities at the marked areas 1 and 2 at 594 and 490 nm, respectively, for 0−180 s. It is obvious that the emission at 594 nm decreases with time. By contrast, the emission at 490 nm increases progressively, indicating the continuous release of [Ca2+]i from the Ca2+-storing organelles. Figure 4f displays the plots of F490/F594 versus time at areas 1 and 2. F490/F594 increases steadily with time until they reach the plateau at ca. 160 s. These results imply that the release of [Ca2+]i from the organelles takes about 3 min, which is in complete agreement with the literature.28−30 In addition, the F490/F594 curves at area 1 and 2 behave similarly, inferring that the ratiometric measurement is very reliable and reproducible regardless of the detection spots in the images. Our work has successfully demonstrated that [Ca2+]i waves in living cells can be real-time monitored by using the proposed Ca2+ probe in conjunction with a CLSM. In conclusion, we have successfully synthesized a probe OBO based on the ICT process. This probe displays high selectivity for Ca2+ operated under ratiometric emission measurement. Its large Stokes shift of 189 nm and large Kd (2.25 ± 0.47 μM) demonstrate its special trait and application as a Ca2+ probe to monitor [Ca2+]i in the resting and dynamic states of living cells. 8029

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