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Bispyrene-Fluorescein Hybrid Based FRET Cassette: A Convenient Platform toward Ratiometric Time-Resolved Probe for Bioanalytical Applications Yongxiang Wu, Xiaobing Zhang, Junbin Li, Cui-Cui Zhang, Hao Liang, Guojiang Mao, Li-Yi Zhou, Weihong Tan, and Ruqin Yu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac502863m • Publication Date (Web): 22 Sep 2014 Downloaded from http://pubs.acs.org on September 25, 2014

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Analytical Chemistry

Bispyrene-Fluorescein Hybrid Based FRET Cassette: A Convenient Platform toward Ratiometric Time-Resolved Probe for Bioanalytical Applications Yong-Xiang Wu, Xiao-Bing Zhang,* Jun-Bin Li, Cui-Cui Zhang, Hao Liang, Guo-Jiang Mao, LiYi Zhou, Weihong Tan, Ru-Qin Yu Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, College of Biology, Hunan University, Changsha 410082. * To whom correspondence should be addressed. E-mail: [email protected]

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ABSTRACT: Pyrene excimer possesses large Stokes shift and long fluorescence lifetime, and has been widely applied in developing time-resolved biosensing systems to solve the autofluorescence interference problems in biological samples. However, only a few of pyrene excimer-based small molecular probes have been reported so far. Ratiometric probes, on the other hand, can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions by a built-in correction of the dual emission bands, but are ineffective for endogenous autofluorescence in biosystems. In this work, by combining the advantages of time-resolved fluorescence technique with ratiometric probe, we reported a bispyrene-fluorescein hybrid FRET cassette (PF) as a novel ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The probe PF showed a fast, highly selective and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffered solution. By employing time-resolved fluorescence technique, the pH induced fluorescence signal of probe PF can be well discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds. It has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since many fluorescein-based fluorescence probes have been developed, our strategy might find wide applications in design ratiometric time-resolved probes for detection of various bio-related targets.


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INTRODUCTION Simple and direct detection or monitoring of significant biological species in complex biological samples is important for understanding their physiological and pathological functions, validating disease biomarkers and diagnosing disease in its early stage.1 Due to its high sensitivity, fast analysis with spatial resolution for providing in situ and real-time information, and nondestructive sample preparation, the fluorescent probe method seems to be an ideal candidate for both detection and monitoring of biomolecules in various biosamples.2 As a consequence, in the past two decades, quite a few fluorescent probes have been developed for various bio-related targets (such as physiological pH values) based on target-triggered fluorescence intensity changes.3 These single emission-based probes usually tend to be affected by a variety of factors such as instrumental efficiency and environmental conditions, as well as the concentration of probe molecule. These interferences can be eliminated by employing ratiometric fluorescent probes,4 which allow the measurement of changes of the intensity ratio at two emission bands to provide built-in correction for the above-mentioned environmental effects. Several strategies, including internal charge transfer (ICT)4a-c and fluorescence resonance energy transfer (FRET),4d-f have been adopted to design ratiometric probes. Among them, FRET strategy could provide moderate resolution of the two emission bands and has been widely applied in designing ratiometric probes for bioanalyitcal applications. However, for application in biosystems, these probes suffer from interference of autofluorescence (fluorescence emitted naturally by a biological substance) from biological environments,5 which might result in decreased detection sensitivity, or even lose the response signal in the background signal. To diminish the interference from biological autofluorescence, several near infrared fluorescence probes have also been developed for intracellular pH measurements.

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Unfortunately, these probes usually could not achieve ratiometric response, and tend to be affected by a variety of environmental factors.


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Pyrene excimer possesses several unique and excellent properties such as large Stokes shift (∼130 nm) and long fluorescence lifetime (∼40 ns),6 which make pyrene excimer-based sensing systems could efficiently eliminate the endogenous autofluorescence interference in biological samples (typically has a lifetime in the order of a picosecond to a few nanoseconds) by using time-resolved (TR) fluorescence assay technique. Such biosensing systems have been developed for the detection of biorelated species including nucleic acids,7 proteins,8 small molecules9 and potassium ions.10 However, only a few of pyrene excimer-based small molecular probes have been reported so far,11 which might be ascribed to the difficult design of recognition units for pyrene excimer to induce efficient signal changes.

Scheme 1. (a) Structure and response mechanism of the probe PF for pH. (b) The principle of fluorescent bioassay based on the TR-FRET technique.

In this work, we try to combine the advantages of ratiometric probe with the time-resolved fluorescence assay to design a FRET-based ratiometric time-resolved sensing platform for bioanalysis. 4 ACS Paragon Plus Environment

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As a proof-of-concept, we have designed and synthesized a bispyrene-fluorescein hybrid (PF) FRET system as a ratiometric time-resolved probe for pH detection (Scheme 1). In this FRET system, a bispyrene moiety was served as the energy donor. Fluorescein was chosen as the energy acceptor based on two facts. Firstly, its absorption spectrum matches well with the fluorescence spectrum of pyrenyl excimer (Figure S1), which guarantee a satisfactory energy transfer efficiency for the system. Secondly, by modifying the phenolic hydroxyl group of fluorescein to realize the target-induced structure transfer from the lactone form to the ring-opened form, it has been proven to be easy to design various fluorescence “turn-on” probes for a variety of targets.2, 12 We chose pH as a model biorelated target, as intracellular pH plays a pivotal role in many cellular events, including cell growth and apoptosis,13 ion transport and homeostasis,14 and so on.15 And the disarrangement of the pH within the different organelles may lead to dysfunction of the affected organelle and ultimately to a diseased state.16 The probe PF showed a fast, highly selective and reversible ratiometric fluorescence response to pH in a wide range from 3.0 to 10.0 in buffer solution. By employing time-resolved fluorescence technique, the pH induced fluorescence signal of probe PF can be well discriminated from biological autofluorescence background, which enables us to detect pH in a range of 4.0-8.0 in cell media within a few seconds without any need of sample clean-up process. The probe exhibits low cytotoxicity, good biocompatibility and intracellular dispersibility, and has also been preliminarily applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. EXPERIMENTAL SECTION Reagents and Apparatus. All chemicals were obtained from commercial suppliers and used without further puriﬁcation. Water used in all experiments was doubly distilled and puriﬁed by a Milli-Q system (Millipore, USA). Solutions of Ca2+, Na+, K+, Mg2+, and Co2+ were prepared from their chloride salts; solutions of Fe3+, Cu2+, and Zn2+ were prepared from their nitrate salts; solutions of Mn2+ were prepared from their acetate salts; solutions of Fe2+ was prepared from their sulfate salts. The cell media, 1640 (Life Technologies, Shanghai, China) supplemented with 10% fetal bovine serum (Life Technologies, ACS Paragon Plus Environment

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Shanghai, China), was used for the detection of pH in the real samples. Thin layer chromatography (TLC) was carried out using silica gel GF254, and column chromatography was conducted over silica gel (300-400 mesh), both of them were obtained from Qingdao Ocean Chemicals (Qingdao, China). LCMS analyses were performed using an Agilent 1100 HPLC/MSD spectrometer. NMR spectra were recorded on a Bruker DRX-400 spectrometer using TMS as an internal standard. All chemical shifts are reported in the standard δ notation of parts per million. The pH was measured with a Mettler-Toledo FE20 pH meter. All fluorescence measurements were carried out on a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ) with both excitation and emission slits set at 5.0 nm. Time-resolved fluorescence spectra were recorded on a FLS900 Fluorescence System (Edinburgh, UK) with excitation at 375nm. UV-Vis absorption spectra were recorded with a Shimadzu UV-2450 spectrophotometer. Fluorescence images of HeLa cells were obtained using an Olympus FV1000 laser confocal microscope (Japan). The synthetic routes of the probe PF are shown in Scheme 2.

H 2N

NH 2

N

NH

H2 N

N

NH2

NH

CH 2Cl2, NaBH 4, EtOH CHO

1 F TC FI , D M N Et 3

O NH

NH

NH

N NH

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O COOH

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OH

PF

Scheme 2. The synthetic routes of the probe PF.

Synthesis of Compound 1. Pyrene-1-carbaldehyde (0.46 g, 2.0 mmol), N1, N1-bis(2-aminoethyl)ethane1, 2-diamine (0.146 g, 1.0 mmol), and an activated NaX zeolite molecular sieve (4 g) were refluxed in CH2Cl2 (100 mL) with magnetic stirring for 2 h under dry N2. The solution was recovered by filtration and concentrated by evaporation. The resulting oil was dissolved in ethanol (100 mL) and stirred with ACS Paragon Plus Environment

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NaBH4 (0.22 g, 6.0 mmol) at 333 K for 2 h and at room temperature for 16 h. The resultant was concentrated by evaporation, dissolved in CH2Cl2, washed with an aqueous NaOH solution (1 mol/L, 25 mL×3), and concentrated by evaporation. The organic layer was separated, dried over anhydrous Na2SO4, and evaporated till dryness. The residue was chromatographed (CH2Cl2/methanol = 20:1 v/v as eluent) to afford pure compound 1 (0.33 g, 57.4%) as a faint yellow solid. 1H NMR (400 MHz, DMSO): δ 8.82 (d, J = 9.0 Hz, 2H), 8.46-8.32 (m, 6H), 8.26 (s, 8H), 8.19-8.00 (m, 2H), 5.77 (s, 4H), 3.02 (m, 12H). ESI-MS [M+]: m/z 574.3. Synthesis of target probe PF. To a solution of compound 1 (53.5 mg, 0.094 mmol) in anhydrous DMF (5 mL) was added fluorescein isothiocyanate (37 mg, 0.094 mmol) and triethyl amine (100 mg, 0.94 mmol). The mixture was stirred for overnight under dry N2 and then a precipitate was formed by the addition of Et2O. This precipitate was washed with water, ethanol and acetone to provide PF (38.9 mg, 43% yeild) as a red powder. 1H NMR (400 MHz, DMSO): 8.25 (d, J = 7.2 Hz, 2H), 8.16 (s, 2H), 8.07 (d, J = 9.2 Hz, 4H), 7.99 (s, 10H), 7.56 (s, 2H), 6.89-6.72 (m, 2H), 6.59 (m, 5H), 5.75 (s, 1H), 4.14 (s, 4H), 2.89 (s, 2H), 2.50 (m, 10H). ESI-MS [M+]: m/z 963.4. Spectrophotometric Experiments. Both the fluorescence and UV-Vis absorption measurement experiments were conduct in H2O/DMSO (95:5, v/v). The steady-state fluorescence emission spectra were recorded at excitation wavelength of 358 nm with emission wavelength range from 370 to 650 nm. A 2 × 10-4 M solution of PF was prepared by dissolving PF in DMSO. A series of standard pH buffers were prepared by mixing 0.1 M HCl and 0.1 M NaOH at varied volume ratios. The solution of PF was prepared by adding 100 µL of the stock solution of PF in a 2 mL volumetric flask, and the solution was diluted to 2 mL with different standard pH buffers and DMSO (H2O/DMSO=95:5, v/v). In the solution thus obtained, the concentrations were 1 × 10-5 M PF with different standard pH buffers. For performance of pH measurement in cell media, a series of different pH cell media were prepared by mixing 0.1 M HCl, 0.1 M NaOH and RPMI 1640 cell media at varied volume ratios. The solution of PF was prepared by adding 1 µL of the stock solution of PF in a 2 mL volumetric flask, and the solution ACS Paragon Plus Environment

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was diluted to 2 mL with different pH cell media and DMSO (H2O/DMSO=95:5, v/v). In the solution thus obtained, the concentrations were 100 nM PF with different pH cell media. The solution was protected from light and kept at 4 °C for further application. Cytotoxicity of Probe PF. In vitro cytotoxicity was measured by performing methyl thiazolyl tetrazolium (MTT) assays19 on the HeLa cells. Cells were seeded into a 96-well cell culture plate at 8×103/well, under 100% humidity, and were cultured at 37 °C and 5% CO2 for 24 h; different concentrations of probe PF (0, 1, 2, 5, 10 ×10-6 M, diluted in RPMI 1640) were then added to the wells. The cells were subsequently incubated for 6 h or 12 h at 37 ˚C under 5% CO2. Then, MTT (10 mL; 5 mg/mL) was added to each well and the plate was incubated for an additional 2 h at 37 °C under 5% CO2. After medium was removed from the well, and DMSO was added into the well. The optical density OD570 value (Abs.) of each well, with background subtraction at 490 nm, was measured by means of a Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT). The following formula was used to calculate the inhibition of cell growth: Cell viability (%) = (mean of Abs. value of treatment group/mean Abs. value of control) ×100%. Cell Cultures and Imaging Experiments. Immediately prior to the imaging experiments, the living Hela cells were washed with phosphate-buffered saline (PBS), incubated with 10 µM probe PF (in the culture medium) for 30 min at 37 °C and then washed with PBS for three times, and imaged. The PFloaded cells were incubated at 37 °C for 30 min in high K+ buffer (30 mM NaCl, 120 mM KCl, 1 mM CaCl2, 0.5 mM MgSO4, 1 mM NaH2PO4, 5 mM glucose, 20 mM HEPES) with various pH values in the presence of 10 µM nigericin. Confocal fluorescence imaging of intracellular different pH in Hela cells was observed under an OLYMPUS FV1000 confocal microscope. Excitation wavelength of laser was 405 nm. Emissions were centered at 460 ± 20 nm and 530 ± 20 nm (double channel). RESULTS AND DISCUSSION


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Optimized Design and Synthesis of the probe PF. In our newly designed ratiometric time-resolved sensing probe for pH detection (Scheme 1), a bispyrene unit was designed as an energy donor, because its fluorescence is insensitive to environmental pH. Moreover, it exists in pyrene excimer form due to the short distance between them, and possesses a large Stokes shift (∼130 nm) and long fluorescence lifetime (∼40 ns), which could efficiently eliminate the endogenous autofluorescence interference in biological samples by using time-resolved fluorescence assay technique. Fluorescein was chosen as an energy acceptor due to its absorption spectrum matches well with steady-state fluorescence emission spectrum of pyrene excimer. Additionally, its fluorescence is pH-sensitive, which makes it possible of the pH-regulated intramolecular FRET process of probe PF. The synthetic routes of the probe PF are shown in Scheme 2. Compound 1, a N-tripodal structural compound consisting of two pyrene moieties and one branched primary amine group, is first synthesized as an intermediate. The probe compound PF was then synthesized by coupling the fluoroscein isothiocyanate (FITC) with compound 1 under base condition. Both of the compounds were characterized using NMR and MS analytical spectroscopic techniques, which agreed well with the proposed structures. (a)

(b) pH=3.0 pH=4.0 pH=5.0 pH=6.0 pH=7.0 pH=8.0 pH=9.0 pH=10.0

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Figure 1. (a) The steady-state fluorescence emission spectra of the ratiometric pH-responsive PF (10 µM) at pH values of 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 in H2O/DMSO (95:5, v/v). λex = 358 nm. (b) pH titration curve of the pH probe PF using the steady-state fluorescence emission intensity ratio I526/I459 as a function of pH. Inset: Change in color (left) and fluorescence (right, excitation at 365 nm) of PF (10 µM) at pH = 3.0 (left) and pH = 10.0 (right).


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The Steady-State Fluorescence Sensing Performance of the Probe PF. The steady-state fluorescence spectral changes of probe PF at a wide range of pH values (3.0-10.0) are shown in Figure 1a. The fluorescence signal changes upon different pH values were completed within a few seconds, which is favorable for real-time tracking of the pH values in biological samples. Increasing pH values give rise to a higher steady-state fluorescence intensity of fluorescein at 526 nm (I526), while the steady-state fluorescence intensity of pyrenyl excimer at 459 nm (I459) decreases concomitantly. The results showed that the FRET between the bispyrene and fluorescein moiety should be responsible for the decreased steady-state intensity of pyrenyl excimer blue emission and the increased steady-state intensity of fluorescein green emission. As seen in Figure 1b, the relative ratio of steady-state fluorescence intensities (I526/I459) increased from 0.26 to 5.82 over the pH range of 3.0-10.0. An easy-to-discern fluorescence color change was observed with different pH values (the inset of Figure 1b), indicating the probe can also serve as a naked-eye probe for pH. The fluorescence quantum yield (QY) of compound 1 (donor) is calculated to be 0.16 with quinine bisulfate in 0.1 M H2SO4 as the standard.6b-d The Förster energy transfer efficiency between pyrenyl excimer and fluorescein in probe PF was calculated to be ∼63.9%, with an estimated r value (the distance between the acceptor and the donor) of 3.57 nm,20 indicating the occurrence of an efficient FRET (see the Supporting Information). Achieving highly selective response to the pH over other potentially competing species coexisting in the sample is a necessity for a fluorescent probe with potential application in biosystems. Therefore, the selectivity experiments of the probe FP were extended to essential metal ions (Ca2+, Co2+, Cu2+, Fe2+, Fe3+, K+, Mg2+, Mn2+, Na+, Zn2+), oxidative-stress associated redox chemicals (including thiols; cysteine (Cys), homocysteine (Hcy), glutathione (GSH)), H2O2 and HClO in aqueous solutions, with results given in Figure 2. No appreciable steady-state relative fluorescence intensity (I526/I459) changes were observed upon the addition of these competing species, which indicates that probe PF could be practically applied for the ratiometric detection of pH without the potential interference from environments. When the solution pH was circularly changed between pH 3.0 and 10.0, one observed


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that steady-state relative fluorescence intensity (I526/I459) of probe PF exhibited repeated changes, indicating a good reversibility of the probe toward pH (Figure 3).

6 5

I526/I459

4 3 2 1

OH -

Na + Zn 2+ Cy s Hc y GS H H 2O HC 2 lO

K+ Mg 2+ Mn 2+

Fe 3+

Cu 2+ Fe 2+

Bla nk Ca 2+ Co 2+

0

Figure 2. Steady-state relative fluorescence intensity (I526/I459) response of PF (10 µM) in the presence of 200 µM of Co2+, Cu2+, Fe2+, Fe3+, Mn2+, Zn2+; 1 mM of Ca2+, Mg2+; 10 mM of K+; 100 mM of Na+; 200 µM of thiols (Cys, Hcy, GSH); 10 mM of H2O2; 10 mM of HClO and 0.1 M NaOH in aqueous solution. λex = 358 nm.

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Figure 3. The reversible steady-state relative fluorescence intensity (I526/I459) changes of PF (10 µM) between pH 3.0 and pH 10.0 in aqueous solution. λex = 358 nm.

The UV-Vis absorption spectra of PF was also investigated at pH values of 3.0-10.0 in H2O/DMSO (95:5, v/v) (Figure S2). At pH values of 3.0, probe PF showed low absorbance at 500 nm, which indicated that the ring closure of the fluorescein cyclolactam in probe PF was the dominant species. With the increasing of pH values, the absorbance centered at 500 nm increased significantly, ACS Paragon Plus Environment

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due to the ring-opening of the fluorescein cyclolactam.21 Meanwhile, two typical pyrene absorbance peaks were observed at 330 and 346 nm, indicating that the bispyrene moiety in probe PF existed in pyrenyl excimer form. As showed in Figure S2, the pyrene absorbance peaks also increased with the increasing pH, and the solution change from colorless to yellow, which could be easily distinguished by naked eye (the inset of Figure 1b). The analysis of UV-Vis absorption data of PF gives a pKa value of 6.65±0.13, which demonstrated that our ratiometric fluorescent probe PF could quantitatively monitor the wide range of pH under physiological conditions (see the Supporting Information). Direct Quantitative Detection of Different pH in Cell Media. The above results demonstrate that the probe PF has excellent selectivity and high sensitivity for detection of pH in simple buffer solution. However, complex biological samples usually contain endogenous components that produce a high autofluorescence background,22 which makes the common fluorescent probes ineffective for assay of biotargets in these systems without sample pretreatment. However, our designed probe PF is based on pyrenyl excimer, which shows much longer lifetimes (> 40 ns) than endogenous autofluorescence (< 5 ns), and it should be easy to discriminate the probe signal from the background signal using timeresolved fluorescence spectroscopy technique. To verify this hypothesis, a cell medium was then used to investigate the feasibility of using this probe for detection of pH in biological samples. First, we recorded the decay curves for PF at pH values of 3.0, 7.0 and 10.0 in H2O/DMSO (95:5, v/v), respectively. Results were shown in Figure S3. With a pH value of 3.0, there is no FRET involved, and we can observe an intensity decay curve for the donor of PF, from which a decay time (or lifetime) of 65.45 ns was determined by fitting the data to a single exponential decay curve. Moreover, the decay time (or lifetime) was determined to be 51.33 ns at pH=7.0 and 48.08 ns at pH=10.0 individually. The decrease in fluorescence emission lifetime of the pyrenyl excimer (donor) with the increasing pH provides additional evidence that in the probe PF the FRET process was turned on by increased pH. This lifetime of probe PF is much longer than the lifetimes of most organic fluorophores and fluorescent components in cell media and cells, which further confirmed the possibility of temporal resolution of the


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fluorescence signal from intense background fluorescence. The steady-state emission spectra of probe PF in buffer solution and 1640 cell media were investigated under different pH values, with results given in Figure 4a and 4b, respectively. In the buffer solution, the probe functioned well, and the characteristic emission peaks of PF was observed at the pH values of 4.0 and 8.0. Unfortunately, in the 1640 cell media, intense background fluorescence from 380 to 650 nm, contributing from some indigenous species in the cell media was observed. This intense background fluorescence buried the signal response from the probe and made the probe signal indistinguishable from the indigenous background fluorescence. This result indicates that steady-state fluorescence measurement is implausible for direct detection of different pH in such a complex biological sample. Time-resolved emission spectra of 100 nM PF in cell media at pH = 4.0 and 8.0 were then carried , respectively (Figure 4c, 4d). Taken over 0 ns decay, the emission spectrum resembled the steady-state emission spectrum, with the fluorescence peak of probe PF being masked by severe background fluorescence from endogenous fluorescence species and scattered light. Because of their short fluorescence lifetimes, the fluorescence and scattered light signal from the cell media decayed rapidly in 20 ns after the excitation pulse. By contrast, the probe PF emission decayed slowly, retaining reasonably high emission intensity even after 20 ns of decay, which allows probe signal to be well separated from the background signal. As a result, the fluorescence emission spectrum of the cell media taken at 20 ns after excitation looked similar to the emission spectra of probe PF in buffer. The temporal separation of background signal from probe signal is evident by comparing the time-resolved fluorescence emission spectrum (20 ns) in Figure 4c, 4d with the steady-state fluorescence emission spectrum of the same sample in Figure 4a, 4b. For steady-state measurement, no resolved peak at 468 nm could be seen when probe PF was in cell media at pH values of 4.0 and 8.0 due to the significant amount of background signal. However, in the time-resolved emission spectrum, which was recorded 20 ns after excitation, the long lifetime emission peaks at 468 nm and 526 nm were well resolved. Thus, this characteristic emission peak could be immediately used to distinguish different pH in biological samples. ACS Paragon Plus Environment

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(a)

(b) Cell Media at pH=4.0 PF in Cell Media at pH=4.0 PF in Buffer at pH=4.0

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Figure 4. Monitoring PF in cell media. Steady-state fluorescence spectra (λex = 358 nm) of cell media at pH = 4.0, 100 nM PF in cell media at pH = 4.0, 100 nM PF in buffer at pH = 4.0 (a); and cell media at pH = 8.0, 100 nM PF in cell media at pH = 8.0, 100 nM PF in buffer at pH = 8.0 (b). Time-resolved fluorescence spectra of 100 nM PF in cell media at pH = 4.0 (c) and 8.0 (d) with different time windows after the excitation pulse, 0 ns and 20 ns. Time-resolved fluorescence emission spectra of 100 nM PF in cell media at pH values of 4.0, 5.0, 6.0, 7.0, and 8.0 with 20 ns time windows (e). pH titration curve of the probe PF using the time-resolved fluorescence emission intensity ratio I526/I468 as a function of pH in cell culture medium (f).

To confirm that our probe PF is feasible for time-resolved qualitative different pH, its timeresolved emission spectra in the cell media with pH values of 4.0, 5.0, 6.0, 7.0, and 8.0 were measured ACS Paragon Plus Environment

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with a delay time of 20 ns (Figure 4e). When the pH values increased, the time-resolved fluorescence intensity of fluorescein at 526 nm (I526) increased and the time-resolved fluorescence intensity of pyrenyl excimer at 468 nm (I468) decreased concomitantly. As seen in Figure 4f, the relative ratio of timeresolved fluorescence intensities (I526/I468) increased from 0.35 to 2.42 over the pH range of 4.0-8.0 in the cell media. Our results demonstrated that the probe PF combined with the time-resolved measurement could be used effectively for direct quantification of pH in complex biological samples. Preliminarily quantitative monitoring of intracellular pH values. To demonstrate the potential use of probe PF in bioimaging applications, we tested the cytotoxicity of PF and ultraviolet light toward HeLa cells, by the reduction activity of methyl thiazolyl tetrazolium (MTT) assay (Figure S4). The viability of untreated cells was assumed to be 100%. Upon incubation of 0-10 ×10-6 M PF for 6 and 12 h, no significant difference in the proliferation of the cells was observed. Specifically, cell viabilities of about 80% even at a high-dose concentration of 1×10-5 M were observed after 6 and 12 h. Furthermore, under the irradiation of ultraviolet light, upon incubation of Hela cells with 10.0 µM PF for 0-30 min, no significant difference in the proliferation of the cells was observed as well. Particularly, cell viabilities of about 75% were observed even under the irradiation of ultraviolet light for 30 min (Figure S4). These data indicated satisfactory biocompatibility of the ratiometric pH fluorescent probe at all dosages, thus, enabling the PF to serve as a potential probe for fluorescence bioimaging.23


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Figure 5. Confocal fluorescence images of probe PF (10.0 µM) in HeLa cells clamped at pH 4 (a-d), 5 (e-h), 6 (i-l), 7 (m-p), and 8 (q-t). The excitation wavelength was 405 nm. First row: bright field image of Hela cells incubated with probe PF (10.0 µM), second row: the fluorescence images were collected at 440-480 nm (pyrenyl excimer), third row: fluorescence image were collected at 510-550 nm (fluorescein), and fourth row: the merge image of second row and third row.

To demonstrate the applicability of probe PF to quantifying intracellular pH, the intracellular imaging experiments were carried for HeLa cells with H+/K+ ionophore nigericin, which is a standard approach for homogenizing the pH of cells and culture medium.24 Similar to other organic probe, our probe PF was also internalized by the cells via endocytosis. The double-channel fluorescence images at (460 ± 20) and (530 ± 20) nm are shown in Figure 5. The intensity of fluorescence from pyrenyl excimer (blue pseudocolor, second row) decreased in cells with the increasing of the pH, whereas the fluorescence from fluorescein (green pseudocolor, third row) increased with the increasing of the pH value. Moreover when intracellular pH were changed from 6 to 7, the color of the Hela cells showed ACS Paragon Plus Environment

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significant changes from blue to green. The important thing is that the fast and drastic blue-to-green color changes of PF in intracellular different pH imaging could be clearly distinguished by using confocal fluorescence microscopy (fourth row); this satisfied the requirement for real-time observations. The relative ratio of intracellular fluorescence intensities (I2/I1) increased from 0.27 to 2.25 with the pH value increased from 4.0 to 8.0 in HeLa cells (Figure S5). Overlay of fluorescence imaging and brightfield images revealed that the fluorescence signals were localized in the cytosol region. Brightfield measurements confirmed that the cells remained viable throughout the imaging experiments. These results indicated that probe PF could quantitatively measure the wide range of intracellular pH values, and monitor pH-dependent signal in living cells.

CONCLUSIONS In summary, we have designed and synthesized a novel bispyrene-fluorescein hybrid FRET cassette PF to serve as a ratiometric time-resolved sensing platform for bioanalytical applications, with pH chosen as a biorelated target. The platform possesses both the advantage of ratiometric fluorescent probe which can eliminate interferences from environmental factors such as instrumental efficiency and environmental conditions, and the advantage of time-resolved probe which can well discriminate the response signal of probe from endogenous autofluorescence background in biological samples. The probe PF exhibited a ratiometric response to a wide range of pH values in both simple buffer solution with the steady-state fluorescence assay and complex cell media by using time-resolved fluorescence measurements without need of any sample pretreatment process. Cellular experimental results demonstrate its low cytotoxicity, good biocompatibility and intracellular dispersibility, and could be applied for ratiometric quantitative monitoring of pH changes in living cells with satisfying results. Since numerous fluorescein-based small molecular probes have been developed to recognize a wide range of targets, our strategy provides a new ratiometric time-resolved sensing platform for direct detection of various targets in biological samples, and could find wide applications in biomedical fields.


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ACKNOWLEDGEMENT This work was supported by the National Key Scientific Program of China (2011CB911000), NSFC (Grants 21325520, 21327009, J1210040, 21177036), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the National Key Natural Science Foundation of China (21135001), the National Instrumentation Program (2011YQ030124), and the Hunan Provincial Natural Science Foundation (Grant 11JJ1002). SUPPORTING INFORMATION AVAILABLE Calculation of energy transfer, pKa of probe PF, time-resolved emission decays data and supplementary figures. This material is available free of charge via the Internet at http://pubs.acs.org.

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