Article pubs.acs.org/ac
Near-Infrared and Naked-Eye Fluorescence Probe for Direct and Highly Selective Detection of Cysteine and Its Application in Living Cells Jianjian Zhang,† Jianxi Wang,† Jiting Liu,‡ Lulu Ning,† Xinyue Zhu,† Bianfei Yu,† Xiaoyan Liu,† Xiaojun Yao,† and Haixia Zhang*,† †
State Key Laboratory of Applied Organic Chemistry, and Key Laboratory of Special Function Materials and Structure Design, Ministry of Education, Lanzhou University, Lanzhou 730000, China ‡ Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources, Ningxia University, Yinchuan 750021, China S Supporting Information *
ABSTRACT: The near-infrared (NIR) fluorescence sensor for rapid, selective, and sensitive detection of cystenine (Cys) is of great importance in both biological and environmental sciences. Herein, we report a specific probe with turn-on fluorescence property, visible color change with naked-eye, and large wavelength shift on UV spectra for highly selective detection of Cys over homocysteine (Hcy) and glutathione (GSH) in both HEPES buffer (10 mM, pH 7.4) and diluted human serum. The probe based on the conjugate addition−cyclization reaction has a low limit of detection to Cys (0.16 μM as NIR fluorescence sensor and 0.13 μM as UV sensor). Kinetic study indicated that the probe has a very rapid response to Cys, owing to the much higher pseudo-first-order reaction constant with Cys (299 M−1 s−1) than with Hcy (1.29 M−1 s−1) or GSH (0.53 M−1 s−1). Upon addition of Cys to a solution of the probe, the color changed from purple to cyan, with the maximum wavelength shifting from 582 to 674 nm in the UV spectrum and a fluorescence emission at 697 nm appearing. It has been successfully applied for determination of Cys in diluted serum and bioimaging of Cys in living cells with low cell toxicity.
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for implementation, cost-effectiveness, real-time detection, noninvasiveness, and good compatibility for biosamples.15−21 Many fluorophore-based fluorescence sensors for the detection of Cys have been developed, including rhodamine,22−24 coumarin,25−28 naphthalimide,29−32 BODIPY,33−36 squaraine,37−39 fluorescein,40−43 and others.44−48 Most of these dyes show emissions and absorption within the ultraviolet or visible range, and are not suitable for bioimaging. Near-infrared (NIR) dyes working at 650−900 nm are greatly important in biological tissue engineering. They possess several merits, such as less damage to living cells, better tissue penetration, and lower background fluorescence with less scattering.49−54 Several fluorescence probes based on near-infrared dyes have been designed. For example, Yoon’s group55 has designed an effective ratiometric cyanine-based NIR probe for Cys over Hcy, and GSH involved the addition of Cys to the acrylic unit to generate the corresponding thioether, and a further intramolecular cyclization to yield the carbonyl derivative. Zhao et al.56 developed a red-emitting Cys probe based on
etection of sulfhydryl-containing amino acids in a physiological system has become an appealing research area nowadays. The sulfhydryl-containing amino acids possess different vital roles in cellular processes.1,2 Cysteine (Cys) and homocysteine (Hcy) are involved in cellular growth, and glutathione (GSH) plays a key role in redox homeostasis.3 The lack of Cys could induce many diseases such as decreased hematopoiesis, leucocyte loss, psoriasis, neurotoxicity, edema, liver damage, and Parkinson’s disease;4−7 the excess of Hcy causes cardiovascular and Alzheimer’s diseases.8−10 Therefore, discrimination of different sulfhydryl-containing amino acids is of great interest and important in biochemistry and biomedicine fields. Some classical methods for determination of biological thiols in the chemical system are achieved by using high-performance liquid chromatography (HPLC) combined with Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid, DTNB),11,12 potentiometry,13 and UV−vis absorption spectrophotometry.14 Although these methods are effective to detect biological thiols in vitro, only a few of them are capable of intracellular detection due to the limitations to in vivo studies. Fluorescence spectroscopy is popular in biological and environmental sciences because of its high sensitivity, fast-response, simplicity © 2015 American Chemical Society
Received: January 29, 2015 Accepted: April 15, 2015 Published: April 15, 2015 4856
DOI: 10.1021/acs.analchem.5b00377 Anal. Chem. 2015, 87, 4856−4863
Article
Analytical Chemistry Scheme 1. Synthesis of CyA
NMR, 7.27 ppm; 13C NMR, 77.0 ppm). The pH values were measured using a digital pH-meter (PHSJ-3F, Leici, Shanghai, China). The fluorescence images of cells were taken using a confocal laser scanning microscope (TCS SP5, Leica, Germany) with an objective lens (×40). Synthesis. Compound 1 was synthesized according to the procedures reported in the literature.61 Synthesis of Compound 2. To a stirred solution of resorcinol (220 mg, 2.0 mmol) in ACN (15 mL) was added K2CO3 (276 mg, 2.0 mmol) at room temperature under nitrogen atmosphere, and the resulting mixture was stirred for 20 min. Then a solution of compound 1 (610 mg, 1.0 mmol) in ACN (10 mL) was added to the above mixture via a syringe, and the reaction mixture was heated at 50 °C for 4 h. Eventually the solvent was evaporated under reduced pressure, and the crude product was purified by flash column chromatography (petroleum ether/CH 2 Cl 2 /CH 3 OH = 25:25:1) on silica gel, affording the desired compound 2 as a blue-green solid (373 mg, yield 73%). Compound 2: mp 189−191 °C; IR (film): νmax = 3429, 2924, 1629, 1580, 1492, 1439, 1361, 1213, 1292, 1258, 1219, 1191, 1143, 1126, 1062, 1030, 978, 913, 804, 727, 563 cm−1; 1 H NMR (400 MHz, CDCl3): δ = 8.04 (d, J = 13.2 Hz, 1H), 7.31 (s, 1H), 7.29−7.23 (m, 2H), 7.20 (d, J = 9.2 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 9.2 Hz, 1H), 6.54 (s, 1H), 5.57 (d, J = 13.6 Hz, 1H), 3.34 (s, 3H), 2.66 (t, J = 6.0 Hz, 2H), 2.62 (d, J = 6.0 Hz, 2H), 1.89 (t, J = 5.8 Hz, 2H), 1.67 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.5, 159.6, 159.1, 143.9, 140.0, 139.5, 131.7, 129.8, 128.1, 127.9, 122.0, 121.9, 116.8, 116.4, 116.0, 107.6, 103.6, 94.1, 47.2, 30.0, 29.7, 28.6, 27.9, 24.5, 21.4 ppm; HRMS (ESI, m/z) Calcd for [C26H26INO2 − I−]: 384.1958, found: 384.1965. Synthesis of CyA. To a stirred solution of compound 2 (51.1 mg, 0.1 mmol) in CH2Cl2 (3 mL) was added triethylamine (Et3N, 28 μL, 0.2 mmol, 2.0 equiv) and acryloyl chloride (16 μL, 0.2 mmol, 2.0 equiv) at 25 °C. After 10 min, the reaction mixture was concentrated under reduced pressure to give crude solid, which was purified by silica gel column chromatography using CH2Cl2/0−10% methanol as eluent to afford desired products. Yield: 48 mg (85%). CyA: mp 147−148 °C; IR (film): νmax = 3548, 3472, 3416, 2922, 1637, 1618, 1145, 1029, 979, 907, 727, 564 cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.64 (d, J = 15.2 Hz, 1H), 7.54 (t, J = 8.0 Hz, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 6.4 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 11.2 Hz, 2H), 7.05 (dd, J = 11.6, 2.0 Hz, 1H), 6.88 (d, J = 15.2 Hz, 1H), 6.69 (d, J = 17.6 Hz, 1H), 6.36 (dd, J = 17.2, 10.8 Hz, 1H), 6.12 (d, J = 10.8 Hz, 1H), 4.21 (s, 3H), 2.87 (t, J = 5.6 Hz, 2H), 2.74 (t, J = 5.2 Hz, 2H), 1.95 (t, J = 5.2 Hz, 2H), 1.80 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δ = 164.0, 163.8, 159.7, 153.0, 152.5, 146.4, 142.1, 141.9, 133.9, 130.7, 130.4, 129.4, 128.1, 128.0, 127.3, 122.2, 119.8, 118.9, 115.9, 113.4, 109.4, 107.0,
styryl-BODIPY. Other NIR probes have been reported recently based on cyanine,57 spirocyclization of benzopyrylium,58 and conjugated dicyanomethylene benzopyran,59 respectively. However, these probes suffer from some drawbacks: (1) low efficiency (response time >30 min); (2) high ratio of organic solvents; and (3) complex synthesis. Hence, designing novel NIR fluorescence probes for selective, speedy, and sensitive detection of Cys is still challenging and essential. Recently we developed a cyanine-based fluorescence probe for biothiols. This probe undergoes biothiol-promoted specific O−S cleavage and subsequent self-immolation through intramolecular 1,6-elimination.60 In addition, we successfully applied the probe for imaging in living cells, in vivo, and in various tissues. However, it cannot distinguish Cys from Hcy and GSH. In this study, we synthesized a novel fluorescence probe (CyA, Scheme 1) with a hemicyanine skeleton. This probe contains an acrylate group as the quenching and recognizing moiety. It proved to be highly selective, rapid (within minutes), and sensitive for determination of Cys over other analytes, including Hcy and GSH. Besides, its color and fluorescence changes have a dramatical response in the event of Cys assault in both aqueous solution and diluted human serum under mild conditions. In addition, this probe was also used for fluorescence imaging of cellular Cys in HeLa cells.
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EXPERIMENTAL SECTION Materials. Cys and GSH were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China). Hcy was obtained from J&K (Beijing, China). Anhydrous dichloromethane (CH2Cl2) was pretreated with CaH2 and fractional distilled before use. Ultrapure water was produced from the ALH-6000-U (Aquapro International Company, USA) purification system. HPLCgrade acetonitrile (ACN) was from Dima Technology (RichmondHill, USA). HeLa cells were obtained as a gift from the Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources of Ningxia University (Yinchuan, China). All other chemicals were obtained from qualified reagent suppliers with analytical reagent grade. Instrumentation. Fluorescence spectra were recorded on a fluorescence spectrometer (RF-5301pc, Japan) with a xenon lamp and 1.0 cm quartz cells at the slits of 5/5 nm. The fluorescence quantum yields were determined on a fluorescence spectrometer FLSP920 (Edinburgh Instruments Ltd., U.K). Absorption spectra were measured on a UV−visible spectrophotometer (TU-1810, China). Mass spectra were measured using Bruker micrOTOF II with ESI mode (America). High resolution mass spectra were measured using a Bruker Daltonics APEX II 47e FT-ICR spectrometer with ESI or APCI positive ion mode (America). NMR spectra were measured using a JEOL 400 MHz instrument (Japan). The chemical shifts (δ) were referenced to residual CHCl3 (1H 4857
DOI: 10.1021/acs.analchem.5b00377 Anal. Chem. 2015, 87, 4856−4863
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Analytical Chemistry
Kinetic Studies.62 The reaction of CyA (5 μM) with Cys, Hcy, and GSH in HEPES buffer (10 mM, pH 7.4) at 37 °C was monitored by measuring the fluorescence intensity (E). The apparent rate constant for the reaction was determined by fitting the fluorescence intensity of the samples to the pseudofirst-order equation:
45.9, 29.7, 29.5, 28.0, 24.4, 20.2 ppm; HRMS (ESI, m/z) Calcd for [C29H28INO3 − I−]: 438.2064, found: 438.2070. General Procedure for Spectra Measurement. The stock solution of CyA (1.0 mM) was prepared in dimethyl sulfoxide (DMSO). The following solutions (8.0 mM) were prepared in deionized water: amino acids (Cys, Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, and Phe), cations (K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+), H2S, and thiophenol (PhSH). The γ-Golbulins solution (8.0 mM) was prepared in HEPES (10 mM, pH 7.4). Test solutions were prepared by placing 20.0 μL of CyA (1.0 mM), 180.0 μL of DMSO, and an appropriate aliquot of each analyte stock solution into a 5.0 mL centrifugal tube, and diluting the solution to 4.0 mL with HEPES (10 mM, pH 7.4). The resulting solution was shaken well at 37 °C for 5 min, and then the fluorescence and UV absorption spectra were recorded. The absorbance ratio R (λ674/λ582) was measured, and the fluorescence spectrum was recorded with the excitation and emission wavelengths at 670/697 nm. Cell Culture. HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with heatinactivated fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37 °C in a 95% humidity atmosphere under 5% CO2 environment. Confocal Microscope Imaging. The cells were seeded in 35 mm diameter glass-bottomed dishes at a density of 3 × 105 cells per dish in RPMI 1640 medium for 24 h. CyA (10 μM) was added to the cells, and the cells were incubated for 15 min at 37 °C. After being washed with Dulbecco’s phosphatebuffered saline (DPBS) twice to remove free probe CyA, the cells were excited by a laser diode (670 nm) and imaged by using a confocal laser scanning microscope (TCS SP5, Leica, Germany) at 680−710 nm. Two control experiments were performed. In the first control experiment, the cells were pretreated first with N-methylmaleimide (NMM, 1 mM) for 15 min at 37 °C, followed by washing with DPBS twice. Then they were incubated with CyA (10 μM) for another 15 min. In the second control experiment, the cells were pretreated with NMM (1.0 mM) for 15 min at 37 °C and washed with DPBS twice. After that, they were treated with Cys (50 μM) and CyA (10 μM) in succession, each for 15 min. All the cells in the control experiments were washed with DPBS twice before imaging. Cytotoxicity Assay. The cytotoxic effects of CyA, compound 2, and DMSO were determined by MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assays. HeLa cells (1 × 104 cells/well) were placed in a flatbottom 96-well plate in 100 μL culture medium and incubated in 5% CO2 at 37 °C for 24 h incubation. The cells were treated with different concentrations (0−50 μM) of CyA, compound 2, and DMSO, respectively. After 24 h incubation, MTT solution (5.0 mg/mL, HEPES) was added into each well (10 μL/well, 0.5 mg/mL) and the residual MTT solution was removed after 4 h, and then DMSO (100 μL) was added to each well to dissolve the formazan crystals. After shaking for 10 min, the absorbance values of the wells were recorded using a microplate reader at 490 nm. The cytotoxic effects (VR) of CyA, compound 2, and DMSO were assessed using the following equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. The assays were performed in six sets for each concentration.
ln((Emax − Et )/Emax ) = −k′t
(1)
where Et and Emax are the fluorescent intensities at 697 nm at times t and the maximum value obtained during the reaction. k′ is the apparent rate constant. The pseudo-first-order rate constant k (M−1 s−1) was obtained from eq 2, (2)
k′ = kC
where C is the concentration of Cys, Hcy, and GSH.
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RESULTS AND DISCUSSION Synthesis of Probe CyA. Scheme 1 depicts the synthesis route of CyA. First, compound 2 was obtained via a retroKnoevenagel reaction by reacting compound 1 with resorcin in the presence of K2CO3 at 50 °C for 4 h using ACN as solvent. Further treatment of compound 2 with acryloyl chloride in CH2Cl2 containing Et3N gave CyA in 62% yield. The chemical structure of CyA was confirmed by 1H NMR, 13C NMR, FTIR, and HRMS (ESI), as shown in the Supporting Information. General Spectral Properties. The spectral properties of CyA were measured in the H2O/DMSO solution (19:1, v/v, 10 mM HEPES, pH = 7.4) with or without Cys (10.0 equiv). CyA showed a major absorption maximum at 582 nm (ε = 3.90 × 104 M−1 cm−1) and weak fluorescence (Φ = 0.016, Table 1). Table 1. Photophysical Data of CyA and Compound 2a Dye
λabs/nm
εmaxb/×105 M−1 cm−1
λem/nm
Φc
Compound 2 CyA
674 582
0.550 0.390
697 696
0.051 0.016
a
10 mM HEPES, 5% (v/v) DMSO, pH = 7.4. bMolar extinction coefficients. cAbsolute quantum yield. Concentrations of CyA and compound 2 are 5 μM. λex = 670 nm.
The absorbance at 582 nm decreased significantly upon addition of Cys (50 μM) to a solution of CyA (5 μM), and a new absorption band centered at 674 nm appeared and increased significantly. The resulting UV spectrum was similar to that of compound 2 (the expected product, λmax = 674 nm, ε = 5.50 × 104 M−1 cm−1). The large wavelength shift led to a visible color change from purple to cyan after addition of Cys (0, 0.25, 1.0, 2.0, 5.0, 7.5, 10.0, 20.0, 25.0, 50.0 μM) to CyA (5 μM), as shown in Figure S1. Meanwhile, the fluorescence signal of CyA displayed noticeable changes (Figure 1b). The emission intensity at 697 nm was enhanced greatly within a few minutes upon adding Cys (10.0 equiv), and the resulting fluorescence spectrum is the same as that of compound 2 (Φ = 0.051) exactly in both the emission intensity and the emission band. Specificity of the Probe. The responses of CyA to 28 substances existing widely in serum were characterized, including 16 amino acids (Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, and Phe), 9 cations (K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+), H2S, PhSH, and γ-Golbulins (a common protein in serum). No obvious changes were observed in the spectra of CyA (Figure 2a and Figure S3, S4). The significant increase of the emission intensity at 697 nm along with the clear color change only corresponded to the 4858
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Figure 1. (a) Absorption and (b) fluorescence emission spectra of compound 2 (5 μM, black line) and CyA (5 μM), before (red line) and after reacting with Cys (50 μM, green line) in H2O/DMSO solution (19:1, v/v, 10 mM HEPES, pH = 7.4), λex = 670 nm.
Figure 2. (a) Fluorescence response of CyA (5 μM) to various analytes (50 μM). Each spectrum was recorded at 5 min after addition of the analytes (Cys, Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, Phe, H2S, PhSH, K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+, and γ-Golbulins). (b) Time-dependent fluorescence changes of CyA (5 μM) upon addition of Cys (50 μM).
Figure 3. (a) Fluorescence titration of CyA (5 μM) upon addition of Cys (0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 40.0, 50.0, 75.0, 100.0, 150.0 μM). Each spectrum was recorded at 5 min after the addition of Cys. (b) Inset: A linear correlation between emission intensities and concentrations of Cys.
addition of Cys to the CyA (Figure S5). The results indicated that the sensing of Cys was hardly influenced by these analytes. Moreover, the response time of Cys was much shorter than those of GSH and Hcy (Figure 2b and Figure S6a). Kinetic analysis (Figure S6b-d) revealed that the apparent rate constant k′ for the reaction of CyA with Cys was 0.8984 min−1, while with Hcy and GSH they were only 0.0039 and 0.0016 min−1, respectively. The pseudo-first-order rate constant k for the reaction of CyA with Cys was 299 M−1 s−1, while with Hcy and GSH it was only 1.29 and 0.53 M−1 s−1, respectively. Based on these results, we confirmed that the probe CyA can detect Cys specifically over Hcy and GSH within a few minutes. The reaction between CyA and Cys was faster than with Hcy due to
the intramolecular attack by the amino group via a sevenmembered ring intermediate for Cys being much more favored than via an eight-membered ring for Hcy. Spectral Response of CyA to Cys. The response ability of CyA to Cys was investigated. As expected, upon addition of Cys, a gradual increase of fluorescence intensity was observed in Figure 3a. The changes in fluorescence titration spectra terminated when the concentration of Cys reached 40 μM and the emission intensity became 10 times higher than that in the absence of Cys. In addition, a good linearity was obtained over the concentration range of 0−25.0 μM for Cys. The regression equation was y = 34.735 + 8.656 × [Cys] with a linear coefficient R of 0.9932 and a standard deviation σ of 0.46. The 4859
DOI: 10.1021/acs.analchem.5b00377 Anal. Chem. 2015, 87, 4856−4863
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Analytical Chemistry limit of detection (LOD, S/N = 3) was calculated as 0.16 μM (Figure 3b). Similarly, UV titration was also accomplished under the same conditions (Figure S2); a good linearity was obtained with σ as 0.03 and LOD as 0.13 μM. This indicates clearly that CyA can be used as both ratiometric UV sensor and NIR sensor for rapid quantitative analysis of Cys. Study on Reaction Mechanism. The acrylate group in CyA has been used as an efficient reaction site for Cys since being reported by Ning et al.63−65 The mechanism of CyA responding to Cys was based on conjugated addition of Cys to an α,β-unsaturated carbonyl moiety, which generated the intermediate thioether, and intramolecular cyclization giving the desired lactam 3 and compound 2 (Scheme 2). In order to
by the change of chromatographic peaks (Figure S8). The decrease of CyA signal was observed after mixing different amounts of Cys, while the signal ascribed to compound 2 appeared and increased. In addition, the mass spectrometry analysis of CyA treated with Cys (1.0, 5.0 equiv) in HEPES buffer also illustrated the formation of the lactam 3 (m/z 176.0657) and the expected compound 2 (m/z 384.1595) (See Figure S9). To gain better insight into the molecular orbital, the structures of probe CyA and compound 2 were optimized and their frontier molecular orbital energies were calculated using a suit of Gaussian 09 programs [density functional theory (DFT) at the B3LYP/6-311G(d, p) level].51,66 As shown in Figure 4, the indolium moiety was coplanar and conjugated with the 2,3-dihydro-1H-xanthene core in compound 2, which led to a fluorescence emission. In addition, the π electrons of compound 2 were mainly located on the whole π-conjugated 2,3-dihydro-1H-xanthene-indolium skeleton on both the LUMO and the HOMO. Moreover, the π electrons on the HOMO of CyA were mainly located on the 2,3-dihydro-1Hxanthene-indolium skeleton, while on the LUMO of CyA they were mainly distributed in the acryloyl group. The LUMO and HOMO levels support the possible photoinduced electron transfer (PET) process in CyA. The electron transfer from the 2,3-dihydro-1H-xanthene-indolium framework (PET donor) to the acryloyl group (PET acceptor) weakened the fluorescence of the primordial fluorophore. This corresponds to the “turnoff” state. The PET process disappeared in compound 2, and the fluorescence was “switched on”. The energy gaps (HOMO − LUMO) of CyA and compound 2 were calculated as 2.02 and 2.77 eV, respectively. The theory calculations are in agreement with the experimental results, which rationalize the PET process. Application. To verify the possibility of CyA for the physiological detection of Cys, the fluorescence responses of CyA were compared at different pH values with or without Cys (Figure S7). It was found that the fluorescence intensities remained nearly the same for CyA at pH ranging from 4.0 to 8.0, and slightly increased at pH from 8.0 to 10.0. However, it
Scheme 2. Proposed Response Mechanism of CyA to Cys
further confirm the sensing mechanism, HPLC analysis was performed. The deprotonation of compound 2 was released from the reaction between CyA and Cys. This was supported
Figure 4. Density functional theory (DFT) optimized structures and frontier molecular orbitals (MOs) of (A) CyA and (B) compound 2. Calculations were based on ground state geometry by DFT at the B3LYP/6-311G (d, p)/level using Gaussian 09 in water. In the ball-and-stick model, carbon, oxygen, and nitrogen atoms are colored in gray, red, and blue, respectively. 4860
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Figure 5. Confocal microscope images of probe CyA in HeLa cells co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify cell nuclei (blue dots). CyA: excitation 670 nm and emission 680−710 nm. (A) control; (B) HeLa cells incubated with CyA (10 μM) for 15 min; (C) HeLa cells pretreated with NMM (1 mM) for 15 min and followed by incubation with CyA (10 μM) for 15 min; (D) HeLa cells pretreated with NMM (1 mM) for 15 min and then treated with Cys (50 μM) for 15 min and incubated with CyA (10 μM) for 15 min. (1) Fluorescence images from DAPI; (2) Fluorescence images from CyA; (3) Merge.
(dissolved in DMSO), and DMSO in living HeLa cells for 24 h to investigate the cytotoxicity. As shown in Figure S11, upon exposure to probe CyA and compound 2 with concentrations of 0−10 μM, the cells kept higher viability under the experimental conditions. However, upon loading 25−50 μM of probe CyA and compound 2, a remarkable toxicity was presented, which was just similar to that from DMSO. In summary, the results indicate that the probe CyA and compound 2 are of low cytotoxicity to the cultured cells and possess great potential for biological applications.
shows obvious enhancement in the presence of Cys in a wide pH range from 6.0−10.0. Some probes were reported using higher pH (pH ≥ 9.0),67,68 which was not suitable for detecting Cys under the physiological pH conditions. In other words, CyA works well in the physiological pH region and pH 7.4 was chosen as the experimental condition. Subsequent investigation in a series of diluted human serum samples69 (in HEPES buffer, 10 mM, pH 7.4) was carried out, and the fluorescence response of CyA (5 μM) at 697 nm was recorded. The Cys concentration was calculated from the standard curve in Figure 3 and summarized in Table S1, and the concentration of mercapto compounds in serum was estimated with the fluorescence emission signal at 12 h after the reaction initiated. The results of mercapto were in accordance with those from Ellman’s method (ESI). 70 The Cys concentration is determined as 83.13 μM in human serum. And the total concentration of mercapto compounds is 94.93 μM with the new fluorescence method and 97.63 μM by Ellman’s medthod.70 Furthermore, Cys with different concentrations from 0.5 to 5 μM was added to the diluted human serum (10%), and satisfactory recoveries were obtained (Table S2). Based on the above results, the confocal fluorescence microscope images were investigated. As shown in Figure 5, living HeLa cells were incubated with CyA at 37 °C for 15 min, and outstanding red fluorescence was observed in the cytoplasm. This indicates CyA has better membrane permeability than ACA.28 In the control test, the cells were treated with NMM for 15 min, followed by CyA for 15 min. As a result, remarkable red emission quenching was observed (Figure 5C). Besides, the NMM-pretreated HeLa cells, which were treated by adding Cys and CyA in sequence, showed a significant increase in red fluorescence (Figure 5D). Furthermore, living HeLa cells displayed dependent brightness change while incubated with CyA in different concentrations (Figure S10). The incubated cells showed a red fluorescence, and the fluorescence grew brighter with the concentrations of CyA increasing from 0 to 20 μM. Moreover, we used a conventional MTT assay of CyA (dissolved in DMSO), compound 2
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CONCLUSIONS
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ASSOCIATED CONTENT
We have successfully synthesized a novel dye CyA based on 2,3-dihydro-1H-xanthene-indolium fluorophore, containing an acrylate group as a reaction site. This dye can be applied for direct and selective detection of cysteine. It is found that CyA can offer high-contrast colorimetric and particular NIR fluorescence turn-on means for detection of Cys within 5 min with a low limit of detection in HEPES buffer. Kinetic study indicated that CyA has much higher pseudo-first-order reduction constant with Cys than those of Hcy and GSH. The cell imaging results suggest that the probe CyA could be applied for sensing intracellular Cys with satisfactory cell membrane permeability and low cytotoxicity. Considering its easy preparation, this new probe shows great potential for applications involving detection of biothiols both in vivo and in vitro.
S Supporting Information *
Additional information as noted in the text (Figures S1−S11, Table S1 and S2); 1H NMR, 13C NMR, and HRMS data of CyA and compound 2. This material is available free of charge via the Internet at http://pubs.acs.org. 4861
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 931 8912058. Fax: +86 931 8912582. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Science Foundation of China (no. 21375052). REFERENCES
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Near-Infrared and Naked-Eye Fluorescence Probe for Direct and Highly Selective Detection of Cysteine and Its Application in Living Cells Jianjian Zhang,† Jianxi Wang,† Jiting Liu,‡ Lulu Ning,† Xinyue Zhu,† Bianfei Yu,† Xiaoyan Liu,† Xiaojun Yao,† and Haixia Zhang*,† †
State Key Laboratory of Applied Organic Chemistry, and Key Laboratory of Special Function Materials and Structure Design, Ministry of Education, Lanzhou University, Lanzhou 730000, China ‡ Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources, Ningxia University, Yinchuan 750021, China S Supporting Information *
ABSTRACT: The near-infrared (NIR) fluorescence sensor for rapid, selective, and sensitive detection of cystenine (Cys) is of great importance in both biological and environmental sciences. Herein, we report a specific probe with turn-on fluorescence property, visible color change with naked-eye, and large wavelength shift on UV spectra for highly selective detection of Cys over homocysteine (Hcy) and glutathione (GSH) in both HEPES buffer (10 mM, pH 7.4) and diluted human serum. The probe based on the conjugate addition−cyclization reaction has a low limit of detection to Cys (0.16 μM as NIR fluorescence sensor and 0.13 μM as UV sensor). Kinetic study indicated that the probe has a very rapid response to Cys, owing to the much higher pseudo-first-order reaction constant with Cys (299 M−1 s−1) than with Hcy (1.29 M−1 s−1) or GSH (0.53 M−1 s−1). Upon addition of Cys to a solution of the probe, the color changed from purple to cyan, with the maximum wavelength shifting from 582 to 674 nm in the UV spectrum and a fluorescence emission at 697 nm appearing. It has been successfully applied for determination of Cys in diluted serum and bioimaging of Cys in living cells with low cell toxicity.
D
for implementation, cost-effectiveness, real-time detection, noninvasiveness, and good compatibility for biosamples.15−21 Many fluorophore-based fluorescence sensors for the detection of Cys have been developed, including rhodamine,22−24 coumarin,25−28 naphthalimide,29−32 BODIPY,33−36 squaraine,37−39 fluorescein,40−43 and others.44−48 Most of these dyes show emissions and absorption within the ultraviolet or visible range, and are not suitable for bioimaging. Near-infrared (NIR) dyes working at 650−900 nm are greatly important in biological tissue engineering. They possess several merits, such as less damage to living cells, better tissue penetration, and lower background fluorescence with less scattering.49−54 Several fluorescence probes based on near-infrared dyes have been designed. For example, Yoon’s group55 has designed an effective ratiometric cyanine-based NIR probe for Cys over Hcy, and GSH involved the addition of Cys to the acrylic unit to generate the corresponding thioether, and a further intramolecular cyclization to yield the carbonyl derivative. Zhao et al.56 developed a red-emitting Cys probe based on
etection of sulfhydryl-containing amino acids in a physiological system has become an appealing research area nowadays. The sulfhydryl-containing amino acids possess different vital roles in cellular processes.1,2 Cysteine (Cys) and homocysteine (Hcy) are involved in cellular growth, and glutathione (GSH) plays a key role in redox homeostasis.3 The lack of Cys could induce many diseases such as decreased hematopoiesis, leucocyte loss, psoriasis, neurotoxicity, edema, liver damage, and Parkinson’s disease;4−7 the excess of Hcy causes cardiovascular and Alzheimer’s diseases.8−10 Therefore, discrimination of different sulfhydryl-containing amino acids is of great interest and important in biochemistry and biomedicine fields. Some classical methods for determination of biological thiols in the chemical system are achieved by using high-performance liquid chromatography (HPLC) combined with Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid, DTNB),11,12 potentiometry,13 and UV−vis absorption spectrophotometry.14 Although these methods are effective to detect biological thiols in vitro, only a few of them are capable of intracellular detection due to the limitations to in vivo studies. Fluorescence spectroscopy is popular in biological and environmental sciences because of its high sensitivity, fast-response, simplicity © 2015 American Chemical Society
Received: January 29, 2015 Accepted: April 15, 2015 Published: April 15, 2015 4856
DOI: 10.1021/acs.analchem.5b00377 Anal. Chem. 2015, 87, 4856−4863
Article
Analytical Chemistry Scheme 1. Synthesis of CyA
NMR, 7.27 ppm; 13C NMR, 77.0 ppm). The pH values were measured using a digital pH-meter (PHSJ-3F, Leici, Shanghai, China). The fluorescence images of cells were taken using a confocal laser scanning microscope (TCS SP5, Leica, Germany) with an objective lens (×40). Synthesis. Compound 1 was synthesized according to the procedures reported in the literature.61 Synthesis of Compound 2. To a stirred solution of resorcinol (220 mg, 2.0 mmol) in ACN (15 mL) was added K2CO3 (276 mg, 2.0 mmol) at room temperature under nitrogen atmosphere, and the resulting mixture was stirred for 20 min. Then a solution of compound 1 (610 mg, 1.0 mmol) in ACN (10 mL) was added to the above mixture via a syringe, and the reaction mixture was heated at 50 °C for 4 h. Eventually the solvent was evaporated under reduced pressure, and the crude product was purified by flash column chromatography (petroleum ether/CH 2 Cl 2 /CH 3 OH = 25:25:1) on silica gel, affording the desired compound 2 as a blue-green solid (373 mg, yield 73%). Compound 2: mp 189−191 °C; IR (film): νmax = 3429, 2924, 1629, 1580, 1492, 1439, 1361, 1213, 1292, 1258, 1219, 1191, 1143, 1126, 1062, 1030, 978, 913, 804, 727, 563 cm−1; 1 H NMR (400 MHz, CDCl3): δ = 8.04 (d, J = 13.2 Hz, 1H), 7.31 (s, 1H), 7.29−7.23 (m, 2H), 7.20 (d, J = 9.2 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.83 (d, J = 7.6 Hz, 1H), 6.76 (d, J = 9.2 Hz, 1H), 6.54 (s, 1H), 5.57 (d, J = 13.6 Hz, 1H), 3.34 (s, 3H), 2.66 (t, J = 6.0 Hz, 2H), 2.62 (d, J = 6.0 Hz, 2H), 1.89 (t, J = 5.8 Hz, 2H), 1.67 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δ = 165.5, 159.6, 159.1, 143.9, 140.0, 139.5, 131.7, 129.8, 128.1, 127.9, 122.0, 121.9, 116.8, 116.4, 116.0, 107.6, 103.6, 94.1, 47.2, 30.0, 29.7, 28.6, 27.9, 24.5, 21.4 ppm; HRMS (ESI, m/z) Calcd for [C26H26INO2 − I−]: 384.1958, found: 384.1965. Synthesis of CyA. To a stirred solution of compound 2 (51.1 mg, 0.1 mmol) in CH2Cl2 (3 mL) was added triethylamine (Et3N, 28 μL, 0.2 mmol, 2.0 equiv) and acryloyl chloride (16 μL, 0.2 mmol, 2.0 equiv) at 25 °C. After 10 min, the reaction mixture was concentrated under reduced pressure to give crude solid, which was purified by silica gel column chromatography using CH2Cl2/0−10% methanol as eluent to afford desired products. Yield: 48 mg (85%). CyA: mp 147−148 °C; IR (film): νmax = 3548, 3472, 3416, 2922, 1637, 1618, 1145, 1029, 979, 907, 727, 564 cm−1; 1H NMR (400 MHz, CDCl3): δ = 8.64 (d, J = 15.2 Hz, 1H), 7.54 (t, J = 8.0 Hz, 2H), 7.49 (d, J = 7.6 Hz, 1H), 7.45 (d, J = 6.4 Hz, 1H), 7.41 (d, J = 8.0 Hz, 1H), 7.12 (d, J = 11.2 Hz, 2H), 7.05 (dd, J = 11.6, 2.0 Hz, 1H), 6.88 (d, J = 15.2 Hz, 1H), 6.69 (d, J = 17.6 Hz, 1H), 6.36 (dd, J = 17.2, 10.8 Hz, 1H), 6.12 (d, J = 10.8 Hz, 1H), 4.21 (s, 3H), 2.87 (t, J = 5.6 Hz, 2H), 2.74 (t, J = 5.2 Hz, 2H), 1.95 (t, J = 5.2 Hz, 2H), 1.80 (s, 6H) ppm; 13C NMR (100 MHz, CDCl3): δ = 164.0, 163.8, 159.7, 153.0, 152.5, 146.4, 142.1, 141.9, 133.9, 130.7, 130.4, 129.4, 128.1, 128.0, 127.3, 122.2, 119.8, 118.9, 115.9, 113.4, 109.4, 107.0,
styryl-BODIPY. Other NIR probes have been reported recently based on cyanine,57 spirocyclization of benzopyrylium,58 and conjugated dicyanomethylene benzopyran,59 respectively. However, these probes suffer from some drawbacks: (1) low efficiency (response time >30 min); (2) high ratio of organic solvents; and (3) complex synthesis. Hence, designing novel NIR fluorescence probes for selective, speedy, and sensitive detection of Cys is still challenging and essential. Recently we developed a cyanine-based fluorescence probe for biothiols. This probe undergoes biothiol-promoted specific O−S cleavage and subsequent self-immolation through intramolecular 1,6-elimination.60 In addition, we successfully applied the probe for imaging in living cells, in vivo, and in various tissues. However, it cannot distinguish Cys from Hcy and GSH. In this study, we synthesized a novel fluorescence probe (CyA, Scheme 1) with a hemicyanine skeleton. This probe contains an acrylate group as the quenching and recognizing moiety. It proved to be highly selective, rapid (within minutes), and sensitive for determination of Cys over other analytes, including Hcy and GSH. Besides, its color and fluorescence changes have a dramatical response in the event of Cys assault in both aqueous solution and diluted human serum under mild conditions. In addition, this probe was also used for fluorescence imaging of cellular Cys in HeLa cells.
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EXPERIMENTAL SECTION Materials. Cys and GSH were purchased from Sangon Biotech. Co., Ltd. (Shanghai, China). Hcy was obtained from J&K (Beijing, China). Anhydrous dichloromethane (CH2Cl2) was pretreated with CaH2 and fractional distilled before use. Ultrapure water was produced from the ALH-6000-U (Aquapro International Company, USA) purification system. HPLCgrade acetonitrile (ACN) was from Dima Technology (RichmondHill, USA). HeLa cells were obtained as a gift from the Key Lab of Ministry of Education for Protection and Utilization of Special Biological Resources of Ningxia University (Yinchuan, China). All other chemicals were obtained from qualified reagent suppliers with analytical reagent grade. Instrumentation. Fluorescence spectra were recorded on a fluorescence spectrometer (RF-5301pc, Japan) with a xenon lamp and 1.0 cm quartz cells at the slits of 5/5 nm. The fluorescence quantum yields were determined on a fluorescence spectrometer FLSP920 (Edinburgh Instruments Ltd., U.K). Absorption spectra were measured on a UV−visible spectrophotometer (TU-1810, China). Mass spectra were measured using Bruker micrOTOF II with ESI mode (America). High resolution mass spectra were measured using a Bruker Daltonics APEX II 47e FT-ICR spectrometer with ESI or APCI positive ion mode (America). NMR spectra were measured using a JEOL 400 MHz instrument (Japan). The chemical shifts (δ) were referenced to residual CHCl3 (1H 4857
DOI: 10.1021/acs.analchem.5b00377 Anal. Chem. 2015, 87, 4856−4863
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Analytical Chemistry
Kinetic Studies.62 The reaction of CyA (5 μM) with Cys, Hcy, and GSH in HEPES buffer (10 mM, pH 7.4) at 37 °C was monitored by measuring the fluorescence intensity (E). The apparent rate constant for the reaction was determined by fitting the fluorescence intensity of the samples to the pseudofirst-order equation:
45.9, 29.7, 29.5, 28.0, 24.4, 20.2 ppm; HRMS (ESI, m/z) Calcd for [C29H28INO3 − I−]: 438.2064, found: 438.2070. General Procedure for Spectra Measurement. The stock solution of CyA (1.0 mM) was prepared in dimethyl sulfoxide (DMSO). The following solutions (8.0 mM) were prepared in deionized water: amino acids (Cys, Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, and Phe), cations (K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+), H2S, and thiophenol (PhSH). The γ-Golbulins solution (8.0 mM) was prepared in HEPES (10 mM, pH 7.4). Test solutions were prepared by placing 20.0 μL of CyA (1.0 mM), 180.0 μL of DMSO, and an appropriate aliquot of each analyte stock solution into a 5.0 mL centrifugal tube, and diluting the solution to 4.0 mL with HEPES (10 mM, pH 7.4). The resulting solution was shaken well at 37 °C for 5 min, and then the fluorescence and UV absorption spectra were recorded. The absorbance ratio R (λ674/λ582) was measured, and the fluorescence spectrum was recorded with the excitation and emission wavelengths at 670/697 nm. Cell Culture. HeLa cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with heatinactivated fetal bovine serum (10%), penicillin (100 U/mL), and streptomycin (100 U/mL) at 37 °C in a 95% humidity atmosphere under 5% CO2 environment. Confocal Microscope Imaging. The cells were seeded in 35 mm diameter glass-bottomed dishes at a density of 3 × 105 cells per dish in RPMI 1640 medium for 24 h. CyA (10 μM) was added to the cells, and the cells were incubated for 15 min at 37 °C. After being washed with Dulbecco’s phosphatebuffered saline (DPBS) twice to remove free probe CyA, the cells were excited by a laser diode (670 nm) and imaged by using a confocal laser scanning microscope (TCS SP5, Leica, Germany) at 680−710 nm. Two control experiments were performed. In the first control experiment, the cells were pretreated first with N-methylmaleimide (NMM, 1 mM) for 15 min at 37 °C, followed by washing with DPBS twice. Then they were incubated with CyA (10 μM) for another 15 min. In the second control experiment, the cells were pretreated with NMM (1.0 mM) for 15 min at 37 °C and washed with DPBS twice. After that, they were treated with Cys (50 μM) and CyA (10 μM) in succession, each for 15 min. All the cells in the control experiments were washed with DPBS twice before imaging. Cytotoxicity Assay. The cytotoxic effects of CyA, compound 2, and DMSO were determined by MTT (3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assays. HeLa cells (1 × 104 cells/well) were placed in a flatbottom 96-well plate in 100 μL culture medium and incubated in 5% CO2 at 37 °C for 24 h incubation. The cells were treated with different concentrations (0−50 μM) of CyA, compound 2, and DMSO, respectively. After 24 h incubation, MTT solution (5.0 mg/mL, HEPES) was added into each well (10 μL/well, 0.5 mg/mL) and the residual MTT solution was removed after 4 h, and then DMSO (100 μL) was added to each well to dissolve the formazan crystals. After shaking for 10 min, the absorbance values of the wells were recorded using a microplate reader at 490 nm. The cytotoxic effects (VR) of CyA, compound 2, and DMSO were assessed using the following equation: VR = A/A0 × 100%, where A and A0 are the absorbance of the experimental group and control group, respectively. The assays were performed in six sets for each concentration.
ln((Emax − Et )/Emax ) = −k′t
(1)
where Et and Emax are the fluorescent intensities at 697 nm at times t and the maximum value obtained during the reaction. k′ is the apparent rate constant. The pseudo-first-order rate constant k (M−1 s−1) was obtained from eq 2, (2)
k′ = kC
where C is the concentration of Cys, Hcy, and GSH.
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RESULTS AND DISCUSSION Synthesis of Probe CyA. Scheme 1 depicts the synthesis route of CyA. First, compound 2 was obtained via a retroKnoevenagel reaction by reacting compound 1 with resorcin in the presence of K2CO3 at 50 °C for 4 h using ACN as solvent. Further treatment of compound 2 with acryloyl chloride in CH2Cl2 containing Et3N gave CyA in 62% yield. The chemical structure of CyA was confirmed by 1H NMR, 13C NMR, FTIR, and HRMS (ESI), as shown in the Supporting Information. General Spectral Properties. The spectral properties of CyA were measured in the H2O/DMSO solution (19:1, v/v, 10 mM HEPES, pH = 7.4) with or without Cys (10.0 equiv). CyA showed a major absorption maximum at 582 nm (ε = 3.90 × 104 M−1 cm−1) and weak fluorescence (Φ = 0.016, Table 1). Table 1. Photophysical Data of CyA and Compound 2a Dye
λabs/nm
εmaxb/×105 M−1 cm−1
λem/nm
Φc
Compound 2 CyA
674 582
0.550 0.390
697 696
0.051 0.016
a
10 mM HEPES, 5% (v/v) DMSO, pH = 7.4. bMolar extinction coefficients. cAbsolute quantum yield. Concentrations of CyA and compound 2 are 5 μM. λex = 670 nm.
The absorbance at 582 nm decreased significantly upon addition of Cys (50 μM) to a solution of CyA (5 μM), and a new absorption band centered at 674 nm appeared and increased significantly. The resulting UV spectrum was similar to that of compound 2 (the expected product, λmax = 674 nm, ε = 5.50 × 104 M−1 cm−1). The large wavelength shift led to a visible color change from purple to cyan after addition of Cys (0, 0.25, 1.0, 2.0, 5.0, 7.5, 10.0, 20.0, 25.0, 50.0 μM) to CyA (5 μM), as shown in Figure S1. Meanwhile, the fluorescence signal of CyA displayed noticeable changes (Figure 1b). The emission intensity at 697 nm was enhanced greatly within a few minutes upon adding Cys (10.0 equiv), and the resulting fluorescence spectrum is the same as that of compound 2 (Φ = 0.051) exactly in both the emission intensity and the emission band. Specificity of the Probe. The responses of CyA to 28 substances existing widely in serum were characterized, including 16 amino acids (Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, and Phe), 9 cations (K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+), H2S, PhSH, and γ-Golbulins (a common protein in serum). No obvious changes were observed in the spectra of CyA (Figure 2a and Figure S3, S4). The significant increase of the emission intensity at 697 nm along with the clear color change only corresponded to the 4858
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Figure 1. (a) Absorption and (b) fluorescence emission spectra of compound 2 (5 μM, black line) and CyA (5 μM), before (red line) and after reacting with Cys (50 μM, green line) in H2O/DMSO solution (19:1, v/v, 10 mM HEPES, pH = 7.4), λex = 670 nm.
Figure 2. (a) Fluorescence response of CyA (5 μM) to various analytes (50 μM). Each spectrum was recorded at 5 min after addition of the analytes (Cys, Hcy, GSH, Gly, Ser, Val, Leu, Tyr, His, Trp, Arg, Glu, Pro, Asp, Thr, Asn, Phe, H2S, PhSH, K+, Na+, Ca2+, Mg2+, Cu2+, Zn2+, Al3+, Fe3+, Fe2+, and γ-Golbulins). (b) Time-dependent fluorescence changes of CyA (5 μM) upon addition of Cys (50 μM).
Figure 3. (a) Fluorescence titration of CyA (5 μM) upon addition of Cys (0, 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 4.0, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 40.0, 50.0, 75.0, 100.0, 150.0 μM). Each spectrum was recorded at 5 min after the addition of Cys. (b) Inset: A linear correlation between emission intensities and concentrations of Cys.
addition of Cys to the CyA (Figure S5). The results indicated that the sensing of Cys was hardly influenced by these analytes. Moreover, the response time of Cys was much shorter than those of GSH and Hcy (Figure 2b and Figure S6a). Kinetic analysis (Figure S6b-d) revealed that the apparent rate constant k′ for the reaction of CyA with Cys was 0.8984 min−1, while with Hcy and GSH they were only 0.0039 and 0.0016 min−1, respectively. The pseudo-first-order rate constant k for the reaction of CyA with Cys was 299 M−1 s−1, while with Hcy and GSH it was only 1.29 and 0.53 M−1 s−1, respectively. Based on these results, we confirmed that the probe CyA can detect Cys specifically over Hcy and GSH within a few minutes. The reaction between CyA and Cys was faster than with Hcy due to
the intramolecular attack by the amino group via a sevenmembered ring intermediate for Cys being much more favored than via an eight-membered ring for Hcy. Spectral Response of CyA to Cys. The response ability of CyA to Cys was investigated. As expected, upon addition of Cys, a gradual increase of fluorescence intensity was observed in Figure 3a. The changes in fluorescence titration spectra terminated when the concentration of Cys reached 40 μM and the emission intensity became 10 times higher than that in the absence of Cys. In addition, a good linearity was obtained over the concentration range of 0−25.0 μM for Cys. The regression equation was y = 34.735 + 8.656 × [Cys] with a linear coefficient R of 0.9932 and a standard deviation σ of 0.46. The 4859
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Analytical Chemistry limit of detection (LOD, S/N = 3) was calculated as 0.16 μM (Figure 3b). Similarly, UV titration was also accomplished under the same conditions (Figure S2); a good linearity was obtained with σ as 0.03 and LOD as 0.13 μM. This indicates clearly that CyA can be used as both ratiometric UV sensor and NIR sensor for rapid quantitative analysis of Cys. Study on Reaction Mechanism. The acrylate group in CyA has been used as an efficient reaction site for Cys since being reported by Ning et al.63−65 The mechanism of CyA responding to Cys was based on conjugated addition of Cys to an α,β-unsaturated carbonyl moiety, which generated the intermediate thioether, and intramolecular cyclization giving the desired lactam 3 and compound 2 (Scheme 2). In order to
by the change of chromatographic peaks (Figure S8). The decrease of CyA signal was observed after mixing different amounts of Cys, while the signal ascribed to compound 2 appeared and increased. In addition, the mass spectrometry analysis of CyA treated with Cys (1.0, 5.0 equiv) in HEPES buffer also illustrated the formation of the lactam 3 (m/z 176.0657) and the expected compound 2 (m/z 384.1595) (See Figure S9). To gain better insight into the molecular orbital, the structures of probe CyA and compound 2 were optimized and their frontier molecular orbital energies were calculated using a suit of Gaussian 09 programs [density functional theory (DFT) at the B3LYP/6-311G(d, p) level].51,66 As shown in Figure 4, the indolium moiety was coplanar and conjugated with the 2,3-dihydro-1H-xanthene core in compound 2, which led to a fluorescence emission. In addition, the π electrons of compound 2 were mainly located on the whole π-conjugated 2,3-dihydro-1H-xanthene-indolium skeleton on both the LUMO and the HOMO. Moreover, the π electrons on the HOMO of CyA were mainly located on the 2,3-dihydro-1Hxanthene-indolium skeleton, while on the LUMO of CyA they were mainly distributed in the acryloyl group. The LUMO and HOMO levels support the possible photoinduced electron transfer (PET) process in CyA. The electron transfer from the 2,3-dihydro-1H-xanthene-indolium framework (PET donor) to the acryloyl group (PET acceptor) weakened the fluorescence of the primordial fluorophore. This corresponds to the “turnoff” state. The PET process disappeared in compound 2, and the fluorescence was “switched on”. The energy gaps (HOMO − LUMO) of CyA and compound 2 were calculated as 2.02 and 2.77 eV, respectively. The theory calculations are in agreement with the experimental results, which rationalize the PET process. Application. To verify the possibility of CyA for the physiological detection of Cys, the fluorescence responses of CyA were compared at different pH values with or without Cys (Figure S7). It was found that the fluorescence intensities remained nearly the same for CyA at pH ranging from 4.0 to 8.0, and slightly increased at pH from 8.0 to 10.0. However, it
Scheme 2. Proposed Response Mechanism of CyA to Cys
further confirm the sensing mechanism, HPLC analysis was performed. The deprotonation of compound 2 was released from the reaction between CyA and Cys. This was supported
Figure 4. Density functional theory (DFT) optimized structures and frontier molecular orbitals (MOs) of (A) CyA and (B) compound 2. Calculations were based on ground state geometry by DFT at the B3LYP/6-311G (d, p)/level using Gaussian 09 in water. In the ball-and-stick model, carbon, oxygen, and nitrogen atoms are colored in gray, red, and blue, respectively. 4860
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Figure 5. Confocal microscope images of probe CyA in HeLa cells co-stained with 4′,6-diamidino-2-phenylindole (DAPI) to identify cell nuclei (blue dots). CyA: excitation 670 nm and emission 680−710 nm. (A) control; (B) HeLa cells incubated with CyA (10 μM) for 15 min; (C) HeLa cells pretreated with NMM (1 mM) for 15 min and followed by incubation with CyA (10 μM) for 15 min; (D) HeLa cells pretreated with NMM (1 mM) for 15 min and then treated with Cys (50 μM) for 15 min and incubated with CyA (10 μM) for 15 min. (1) Fluorescence images from DAPI; (2) Fluorescence images from CyA; (3) Merge.
(dissolved in DMSO), and DMSO in living HeLa cells for 24 h to investigate the cytotoxicity. As shown in Figure S11, upon exposure to probe CyA and compound 2 with concentrations of 0−10 μM, the cells kept higher viability under the experimental conditions. However, upon loading 25−50 μM of probe CyA and compound 2, a remarkable toxicity was presented, which was just similar to that from DMSO. In summary, the results indicate that the probe CyA and compound 2 are of low cytotoxicity to the cultured cells and possess great potential for biological applications.
shows obvious enhancement in the presence of Cys in a wide pH range from 6.0−10.0. Some probes were reported using higher pH (pH ≥ 9.0),67,68 which was not suitable for detecting Cys under the physiological pH conditions. In other words, CyA works well in the physiological pH region and pH 7.4 was chosen as the experimental condition. Subsequent investigation in a series of diluted human serum samples69 (in HEPES buffer, 10 mM, pH 7.4) was carried out, and the fluorescence response of CyA (5 μM) at 697 nm was recorded. The Cys concentration was calculated from the standard curve in Figure 3 and summarized in Table S1, and the concentration of mercapto compounds in serum was estimated with the fluorescence emission signal at 12 h after the reaction initiated. The results of mercapto were in accordance with those from Ellman’s method (ESI). 70 The Cys concentration is determined as 83.13 μM in human serum. And the total concentration of mercapto compounds is 94.93 μM with the new fluorescence method and 97.63 μM by Ellman’s medthod.70 Furthermore, Cys with different concentrations from 0.5 to 5 μM was added to the diluted human serum (10%), and satisfactory recoveries were obtained (Table S2). Based on the above results, the confocal fluorescence microscope images were investigated. As shown in Figure 5, living HeLa cells were incubated with CyA at 37 °C for 15 min, and outstanding red fluorescence was observed in the cytoplasm. This indicates CyA has better membrane permeability than ACA.28 In the control test, the cells were treated with NMM for 15 min, followed by CyA for 15 min. As a result, remarkable red emission quenching was observed (Figure 5C). Besides, the NMM-pretreated HeLa cells, which were treated by adding Cys and CyA in sequence, showed a significant increase in red fluorescence (Figure 5D). Furthermore, living HeLa cells displayed dependent brightness change while incubated with CyA in different concentrations (Figure S10). The incubated cells showed a red fluorescence, and the fluorescence grew brighter with the concentrations of CyA increasing from 0 to 20 μM. Moreover, we used a conventional MTT assay of CyA (dissolved in DMSO), compound 2
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CONCLUSIONS
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ASSOCIATED CONTENT
We have successfully synthesized a novel dye CyA based on 2,3-dihydro-1H-xanthene-indolium fluorophore, containing an acrylate group as a reaction site. This dye can be applied for direct and selective detection of cysteine. It is found that CyA can offer high-contrast colorimetric and particular NIR fluorescence turn-on means for detection of Cys within 5 min with a low limit of detection in HEPES buffer. Kinetic study indicated that CyA has much higher pseudo-first-order reduction constant with Cys than those of Hcy and GSH. The cell imaging results suggest that the probe CyA could be applied for sensing intracellular Cys with satisfactory cell membrane permeability and low cytotoxicity. Considering its easy preparation, this new probe shows great potential for applications involving detection of biothiols both in vivo and in vitro.
S Supporting Information *
Additional information as noted in the text (Figures S1−S11, Table S1 and S2); 1H NMR, 13C NMR, and HRMS data of CyA and compound 2. This material is available free of charge via the Internet at http://pubs.acs.org. 4861
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AUTHOR INFORMATION
Corresponding Author
*Tel.: +86 931 8912058. Fax: +86 931 8912582. E-mail: [email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work was supported by National Science Foundation of China (no. 21375052). REFERENCES
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