Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based

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A Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based on an Apoferritin-Assembled Lanthanide Complex-Rhodamine LRET System Lu Tian, Zhichao Dai, Xiangli Liu, Bo Song, Zhiqiang Ye, and Jingli Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b02347 • Publication Date (Web): 13 Oct 2015 Downloaded from http://pubs.acs.org on October 14, 2015

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Analytical Chemistry

A Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based on an Apoferritin-Assembled Lanthanide Complex-Rhodamine LRET System Lu Tian, Zhichao Dai, Xiangli Liu, Bo Song, Zhiqiang Ye, Jingli Yuan*

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian 116024, China

*

Corresponding author.

Tel./Fax: +86-411-84986042; E-mail: [email protected]

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ABSTRACT: Using apoferritin (AFt) as a carrier, a novel ratiometric luminescence probe based on luminescence resonance energy transfer (LRET) between a Tb3+ complex (PTTA-Tb3+) and a rhodamine derivative (Rh-NO), PTTA-Tb3+@AFt-Rh-NO, has been designed and prepared for the specific recognition and time-gated luminescence detection of nitric oxide (NO) in living samples. In this LRET probe, PTTA-Tb3+ encapsulated in the core of AFt is the energy donor, and Rh-NO, a NO-responsive rhodamine derivative, bound on the surface of AFt is the energy acceptor. The probe only emits strong Tb3+ luminescence because the emission of rhodamine is switched off in the absence of NO. Upon reaction with NO, accompanied by the turn-on of rhodamine emission, the LRET from Tb3+ complex to rhodamine occurs, which results in the remarkable increase and decrease of the long-lived emissions of rhodamine and PTTA-Tb3+, respectively. After the reaction, the intensity ratio of rhodamine emission to Tb3+ emission, I565/I539, is ~24.5-fold increased, and the dose-dependent enhancement of I565/I539 shows a good linearity in a wide concentration range of NO. This unique luminescence response allowed PTTA-Tb3+@AFt-Rh-NO to be conveniently used as a ratiometric probe for the time-gated luminescence detection of NO with I565/I539 as a signal. Taking advantages of high specificity and sensitivity of the probe, as well as its good water-solubility, biocompatibility and cell membrane permeability, PTTA-Tb3+@AFt-Rh-NO was successfully used for the luminescent imaging of NO in living cells and Daphnia magna. The results demonstrated the efficacy of the probe and highlighted it advantages for the ratiometric time-gated luminescence bioimaging application.


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Nitric oxide (NO), one of the significant endogenously generated gaseous signaling molecules, plays vital roles in various physiological processes, such as blood pressure control, growth regulation, cell differentiation, neurotransmission and immune response.1-4 However, the excessive accumulation of NO and its biotransformation products such as peroxynitrite (ONOO-), dinitrogen trioxide (N2O3), and other reactive nitrogen species in organisms will induce the oxidative and nitrosative damage to lipids, DNA and proteins, which are related with many human diseases including Alzheimer’s and Parkinson’s diseases, etc.5-8 Therefore, it is highly desirable to develop selective and sensitive methods for the in vivo detection of NO in complicated biological samples. To date, although a variety of analytical methods for the detection of NO have been established, such as electron paramagnetic resonance spectroscopy,9-10 electrochemistry,11-12 colorimetric,13-14 and chemiluminescence methods,15-21 fluorometric method using NO-responsive fluorescent probes can be considered to be the most appreciated method for the in vivo real-time detection of NO in living samples due to its high selectivity and sensitivity, experimental feasibility, spatial resolution ability, and non-invasive damage to biological specimens.22,23 However, the majority of these fluorescent probes are the probes using fluorescence intensity at a single wavelength as the signal. The fluorescence measurement using these probes is often suffered from the influence of the variations of excitation intensity, probe concentration, sample thickness and microenvironment.23-26 To solve this problem, the design of ratiometric fluorescence probes that enable simultaneous recording of two measurable signals at two different wavelengths in the presence of target analyte is a quite promising approach.24,27 Luminescent lanthanide complexes, especially those of Eu3+ and Tb3+, exhibit several special luminescence properties, including super long luminescence lifetimes, large Stokes shifts and sharp emission profiles, which enable them to be designed as time-gated luminescence probes for the 3 ACS Paragon Plus Environment


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detection of target analyte in complicated biological samples to eliminate the interferences of autofluorescence and scattering lights.28-31 Thus, the combination of ratiometric probe with time-gated probe can be considered to be a more excellent approach for improving the accuracy of luminescence bioassays. However, the rational design of lanthanide complex-based ratiometric luminescence probes is still rather difficult at this moment.32 Among various ratiometric protocols, fluorescence resonance energy transfer (FRET) has been widely adopted as a tool for the design of ratiometric fluorescence probes.33-35 In general, several FRET systems based on coumarin-fluorescein,35 coumarin-rhodamine,36 fluorescein-rhodamine,37,38 and BODIPY-BODIPY dyes39 are easily selected for the design of ratiometric probes. Unfortunately, those ratiometric probes suffer still the interferences of autofluorescence when they are used for bioimaging, which limits their effective application in complicated biological systems. Although some luminescent lanthanide complexes have been used as energy donors in luminescence resonance energy transfer (LRET) applications,40-47 these works mainly explored the applications of LRET for nucleic acid hybridization assays, immunoassays and biological molecular interactions. The LRET-based ratiometric luminescence probes for the time-gated luminescence detection of small bioactive molecules have rarely been investigated. In this work, we constructed a unique LRET-based ratiometric time-gated luminescence probe for NO, PTTA-Tb3+@AFt-Rh-NO, by using a luminescent Tb3+ complex, PTTA-Tb3+, as the energy donor, and a NO-responsive rhodamine derivative, Rh-NO, as the energy acceptor, which were encapsulated in apoferritin and covalently bound on the surface of apoferritin, respectively (Scheme 1). The probe itself emits only the strong Tb3+ luminescence because Rh-NO is a dark-state rhodamine derivative. After reaction with NO, the emission of rhodamine is switched on, so that the LRET from Tb3+ complex to rhodamine occurs, to result in the remarkable increase and decrease of 4 ACS Paragon Plus Environment

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emission intensities at 577 nm (rhodamine emission) and 539 nm (Tb3+ emission) of the probe, respectively. It was found that the emissions of rhodamine and Tb3+ were all long-lived in the LRET system, and the emission intensity ratio of rhodamine/Tb3+, IRh/ITb, showed a good linear correlation to the concentration of NO. These features enabled PTTA-Tb3+@AFt-Rh-NO to be used as a ratiometric time-gated luminescence probe for the detection of NO with IRh/ITb as the signal. To examine the feasibility of the new probe for bioimaging, the PTTA-Tb3+@AFt-Rh-NO-loaded HepG2 cells and Daphnia magna were prepared, and the exogenous NO was imaged on a true-color time-gated luminescence microscope. Scheme 1 shows structures of PTTA-Tb3+ and Rh-NO, and general depiction of the luminescence response of PTTA-Tb3+@AFt-Rh-NO to NO.

EXPERIMENTAL SECTION Materials and Physical Measurements. The ligand PTTA was synthesized according to the previous method.48 5-Carboxytetramethylrhodamine (compound 1) was purchased from Zibo Yunhui

Bio-technology

Co.,

Ltd

(China).

2-(4-Carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (c-PTIO) and apoferritin from equine

spleen

were

purchased

1-Hydroxy-2-oxo-3-(3-amino-propyl)-3-methyl-1-triazene

from (NOC-13,

a

Sigma-Aldrich. NO

donor

with

a

half-lifetime of 13.7 min) was synthesized using a reported method.49 Anhydrous acetonitrile was used after appropriate distillation and purification. Cultured HepG2 cells were obtained from Dalian Medical University. Cultured Daphnia magna were obtained from Professor Jingwen Chen’s group at School of Environmental Science and Technology, Dalian University of Technology. The isotonic saline solution consisting of 140 mM NaCl, 10 mM glucose, and 3.5 mM KCl was prepared in our laboratory. Deionized and distilled water was used throughout. Unless otherwise stated, all chemical 5 ACS Paragon Plus Environment


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materials were purchased from commercial sources and used without further purification. 1

H NMR spectra were measured on a Bruker Avance spectrometer (400 MHz). Mass spectra

were recorded on a HP1100 LC/MSD MS spectrometer. Time-gated luminescence spectra were measured on a Perkin-Elmer LS 50B luminescence spectrometer with the settings of delay time, 0.1 ms; gate time, 0.1 ms; cycle time, 20 ms; excitation slit, 10 nm; and emission slit, 7 nm. Luminescence lifetimes were measured on an Edinburgh OB920FP Fluorescence and Phosphorescence Lifetime Spectrometer. All bright-field and luminescence imaging measurements were carried out on a laboratory-use true color time-gated luminescence microscope.50,51 Synthesis of Rh-NO. The reaction pathway for the synthesis of Rh-NO is shown in Scheme 2. The experimental details are described as follows. Synthesis of Compound 2. To a solution of compound 1 (200 mg, 0.47 mmol) in 30 mL of anhydrous CH3CN were added N,N’-dicyclohexylcarbodiinide (DCC, 100 mg, 0.47 mmol) and dimethylaminopyridine (DMAP, 4.2 mg, 0.028 mmol). The solution was stirred for 3 h at room temperature, and then mono-t-Boc-piperazine (154 mg, 0.47 mmol) in 10 mL of anhydrous CH3CN was added. After stirring for another 3 h, the solution was filtered, and the filtrate was evaporated. The residue was purified by silica gel column chromatography, using acetonitrile-water (10:1, v/v) as the eluent. Compound 2 was obtained as a red solid (160 mg, 56.8% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) = 1.49 (s, 9H), 3.00 (s, 12H), 3.49-3.79 (m, 8H), 6.40 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 4.0 Hz, 2H), 6.61 (d, J = 12 Hz, 2H), 7.23 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 1H), 8.00 (s, 1H). ESI-MS (m/z): 599.2 [M]+. Synthesis

of

Compound

3.

A

mixture

of

compound

2

(160

mg,

0.27

mmol),

N-hydroxysuccinimide (NHS, 115 mg, 0.81 mmol) and DCC (167 mg, 0.81 mmol) in dry CH2Cl2 (20 mL) was stirred for 3 h at room temperature. After filtering, the filtrate was evaporated. The 6 ACS Paragon Plus Environment

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residue was re-dissolved in 10 mL of dry CH2Cl2, triethylamine (150 µL) and o-phenylenediamine (58 mg, 0.54 mmol) were added. After stirring for 2 h at room temperature, the solvent was evaporated, and the crude product was purified by silica gel column chromatography, using ethyl acetate-petroleum ether (1:3, v/v) as the eluent. Compound 3 was obtained as a white solid (104 mg, 56% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) = 1.49 (s, 9H), 2.95 (s, 12H), 3.40-3.60 (m, 8H), 6.08 (d, J = 8.0 Hz, 1H), 6.31 (s, 2H), 6.37-6.42 (m, 3H), 6.56 (d, J = 4.0 Hz, 1H), 6.70 (d, J = 8 Hz, 2H), 6.93-6.97 (m, 1H), 7.28, (s, 1H), 7.64 (d, J = 4.0 Hz, 1H), 8.02 (s, 1H). ESI-MS (m/z): 689.4 [M + H]+. Synthesis of Rh-NO. Compound 3 (0.19 mmol) was dissolved in 10 mL of CH2Cl2, and then concentrated HCl (120 µL) was added to the solution. After stirring for 50 min, the solvent was evaporated. The residue was re-dissolved in 5.0 mL of CH2Cl2, and triethylamine (TEA, 500 µL) was added. After stirring for another 15 min, the solvent was evaporated, and the crude product was purified by silica gel column chromatography, using CH3CN-H2O (8:1, v/v) as the eluent. Rh-NO was obtained as a white solid (85 mg, 76% yield). 1H NMR (400 MHz, CDCl3): δ (ppm) = 2.95 (s, 12H), 3.47-3.84 (m, 8H), 6.06 (d, J = 8.0 Hz, 1H), 6.30 (s, 2H), 6.36-6.44 (m, 3H), 6.58 (d, J = 8.0 Hz, 1H), 6.68 (d, J = 8.0 Hz, 2H), 6.94-6.98 (m, 1H), 7.28 (s, 1H), 7.65 (d, J = 8.0 Hz, 1H), 8.00 (s, 1H). ESI-MS (m/z): 589.3 [M + H]+. Preparation

of

PTTA-Tb3+@AFt-Rh-NO.

The

preparation

principle

of

PTTA-Tb3+@AFt-Rh-NO is shown in Scheme S1 in Supporting Information. The experimental details are described as follows. Preparation of PTTA-Tb3+@AFt. To a solution of PTTA (6.6 mg, 9.0 µmol) dissolved in 0.4 mL distilled water was added TbCl3·6H2O (3.4 mg, 9.0 µmol) under stirring. After the mixture was stirred for 30 min, an aqueous solution of apoferritin (0.3 mL, 3 × 10-2 µmol) was added (the molar 7 ACS Paragon Plus Environment


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ratio of apoferritin/Tb3+ complex is ~1:300). The pH value of the solution was lowered to 2.0 with concentrated HCl, followed by continuously stirring at this pH for 1 h. Then the pH value was adjusted to 8.5 with 1.0 M NaOH, and the solution was further stirred for 2 h. After centrifugation (12500 rpm) at 4 °C for 20 min to remove trace undissolved products, the supernatant was collected. Preparation of PTTA-Tb3+@AFt-Rh-NO. To the above PTTA-Tb3+@AFt solution were added Rh-NO (13.3 mg, 22.6 µmol) dissolved in 0.3 mL of DMSO and 0.4 mL of 1% glutaraldehyde. The reaction mixture was stirred at 4 °C for 22 h, and then 1.0 mg of NaBH4 was added. The solution was stirred for another 2 h at room temperature. After centrifugation (12500 rpm) at 4 °C for 20 min, the supernatant was dialyzed for 24 h against 3 L of distilled water at 4 °C. The transparent solution of PTTA-Tb3+@AFt-Rh-NO was obtained with a final volume of 4.37 mL. Luminescence Imaging of NO in Living Samples. In this work, two kinds of living samples, cultured HepG2 cells and Daphnia magna, were used for the luminescence imaging measurements. The experimental details are described as follows. HepG2 Cells. The cells were cultured on a glass-bottom culture dish (φ 20 mm) in RPMI-1640 medium, supplemented with 10% fetal bovine serum, 1% penicillin, and 1% streptomycin at 37 °C in a 5% CO2/95% air incubator. After washing twice with the isotonic saline solution, the cells were incubated with the mixture of 925 µL culture medium and 75 µL PTTA-Tb3+@AFt-Rh-NO for 24 h. After washing, the cells were further incubated with the isotonic saline solution containing NOC-13 (1.0 mM) for another 1 h. The cells were washed three times with the isotonic saline solution, and then subjected to the luminescence imaging measurements. The steady-state luminescence imaging measurements were carried out with exposure time of 0.5 ms, and time-gated luminescence imaging measurements were carried out with the conditions of delay time, 33 µs; gate time, 1.0 ms; lamp 8 ACS Paragon Plus Environment

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pulse width, 80 µs, and exposure time, 7 s. Daphnia magna. The newborn Daphnia magna (age < 48 h), cultured in nonchlorinated tap water at 20 oC under cool-white fluorescent light with a 14:10 h light:dark photoperiod, were incubated with the mixture of 0.1 mL PTTA-Tb3+@AFt-Rh-NO and 0.4 mL non-chlorinated tap water for 1 h at 25 °C. After washing, they were further incubated with the non-chlorinated tap water containing NOC-13 (1.0 mM) for another 20 min. The Daphnia magna were washed three times with non-chlorinated tap water, and then subjected to the luminescence imaging measurements with the same conditions as those of cell imaging.

RESULTS AND DISCUSSION Design, Preparation and Characterization of the Probe. As a most convenient approach, FRET mechanism has been widely used for the design of various ratiometric fluorescence probes by using organic dyes as energy donors and acceptors. However, this approach was rarely used for the design of lanthanide complex-based ratiometric time-gated luminescence probes. The main reason maybe is due to the lower emission efficiency of lanthanide complexes. It is quite difficult to incorporate multiple molecules of lanthanide complexes into a conventional small-molecular system, as the number of attachment sites on a small molecule is limited, which is unfavorable when lanthanide complexes are used as energy donors for the design of LRET systems. To achieve this goal, we resorted to use a protein, apoferritin, as the carrier for the design of the LRET-based ratiometric time-gated luminescence probe for NO in this work. Apoferritin, composed of 24 polypeptide subunits with a hollow cage spherical structure having internal and external diameters of 8 and 12 nm, has been used for the preparation of lanthanide complex-based luminescent probes for time-gated luminescence bioimaging detections.52,53 One 9 ACS Paragon Plus Environment


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unique property of apoferritin is its self-assembly property: its polypeptide subunits can dissociate at pH 2.0, and restore to an intact hollow cage structure when the pH of the solution is slowly adjusted to neutral. Using this feature of apoferritin, a luminescent protein, PTTA-Tb3+@AFt, was prepared

by

encapsulating

PTTA-Tb3+

into

apoferritin

through

the

pH-modulated

dissociation-reassembly method. After covalently conjugating Rh-NO to the amino groups on the external surface of PTTA-Tb3+@AFt, the LRET-based ratiometric luminescence probe for NO, PTTA-Tb3+@AFt-Rh-NO, was prepared in this work (Scheme S1 in Supporting Information). After being purified by dialysis, the obtained PTTA-Tb3+@AFt-Rh-NO was characterized by transmission electron microscopy (TEM). As shown in Figure S1 in Supporting Information, the obtained PTTA-Tb3+@AFt-Rh-NO particles are monodisperse, spherical and uniform in size, ~12 nm in diameter, which is well consistent with the native apoferritin, indicating that apoferritin molecules have been self-assembled without significant changes of shape and size during the dissociation-reassembly process. Because the LRET efficiency is greatly depended on the extent of the spectral overlap between the donor emission and acceptor absorption, the emission spectrum of PTTA-Tb3+ and excitation and emission spectra of 5-carboxytetramethylrhodamine were recorded. As shown in Figure 1A, the emission peaks of PTTA-Tb3+ at 487 and 539 nm are well overlapped with the excitation spectrum of 5-carboxytetramethylrhodamine, which satisfies the pre-requirement for the LRET from the Tb3+ complex to rhodamine. It can be observed that the emission of PTTA-Tb3+ is nearly silent at 565 nm (~100-fold lower than that of its emission peak at 539 nm), while that of rhodamine at 565 nm is still strong with ~90% intensity of its emission peak at 577 nm. Thus, the intensity of long-lived emission of rhodamine at 565 nm can be used as the specific signal of LRET when PTTA-Tb3+ and rhodamine are used as the donor-acceptor pair for the LRET measurement, to avoid the effect of the 10 ACS Paragon Plus Environment

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emission of PTTA-Tb3+ at ~580 nm. To examine the practical luminescence response of the probe to NO, time-gated emission spectra of PTTA-Tb3+@AFt-Rh-NO before and after reaction with NO were recorded in 50 mM PBS buffer of pH 7.4. As shown in Figure 1B, the probe itself exhibited only the characteristic time-gated emission spectrum of the Tb3+ complex with a main peak at 539 nm (5D4→7F5) and several side peaks centered at 487, 580 and 617 nm. This is correspondence with the fact that the emission of rhodamine in the probe is switched off. However, after reaction with NO, the emission spectrum of the probe showed remarkable changes with dramatically enhanced emission of rhodamine at 565 nm and obviously declined emission of PTTA-Tb3+ at 539 nm. This phenomenon indicates that: (1) the LRET from PTTA-Tb3+ to rhodamine occurred after PTTA-Tb3+@AFt-Rh-NO was reacted with NO; (2) the emission lifetime of rhodamine in the LRET system was effectively prolonged, which allowed it to be suitable for the time-gated detection. By measuring the lifetimes of the donor’s emission in the absence of acceptor (τD, 331.3 µs, decoded from the luminescence decay curve in Figure S2A in Supporting Information) and the sensitized emission of acceptor (τAD, 73.7 µs, decoded from the luminescence decay curve in Figure S2B in Supporting Information), the LRET efficiency (E) was calculated using the equation54 E = 1 – (τAD/ τD) to be 77.8%, which reveals that the time-gated luminescence response of PTTA-Tb3+@AFt-Rh-NO to NO is highly effective, and this probe could indeed be used as a ratiometric probe for the time-gated luminescence detection of NO with the I565/I539 ratio as a signal. Ratiometric Time-Gated Luminescence Detection of NO Using PTTA-Tb3+@AFt-Rh-NO as a Probe. Initially, to evaluate the performance of PTTA-Tb3+@AFt-Rh-NO as a ratiometric time-gated luminescence probe for the quantitative detection of NO in aqueous media, the time-gated emission spectra of PTTA-Tb3+@AFt-Rh-NO upon reaction with different 11 ACS Paragon Plus Environment


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concentrations of NO (NOC-13 was used as a NO source16) were recorded in 50 mM PBS buffer of pH 7.4. As shown in Figure 2A, upon reaction with different concentrations of NO, the time-gated emission intensities of PTTA-Tb3+@AFt-Rh-NO at 577 nm and 539 nm were gradually increased and decreased, respectively. The inset in Figure 2A shows the variation of the intensity ratio of rhodamine/Tb3+ emissions, I565/I539, as a function of NO concentration. This result shows that the I565/I539 ratio of PTTA-Tb3+@AFt-Rh-NO is increased from 0.0148 to 0.363 with the increase of NO concentration from 4.0 µM to 1.0 mM, which provides a ~24.5-fold contrast window for the detection of NO. Moreover, the dose-dependent enhancement of the I565/I539 ratio showed a good linearity in a wide NO concentration range (Figure 2B). The detection limit for NO, defined as the concentration corresponding to trinal standard deviations of the background signal, was calculated to be 0.74 µM, which demonstrated the feasibility of PTTA-Tb3+@AFt-Rh-NO as a ratiometric probe for the quantitative time-gated luminescence detection of NO at a low micromolar concentration level. To examine the luminescence response specificity of the probe to NO, the I565/I539 ratios of PTTA-Tb3+@AFt-Rh-NO upon reaction with different reactive oxygen/nitrogen species (ROS/RNS) were measured in 50 mM PBS buffer of pH 7.4. As shown in Figure 3, the I565/I539 ratio of PTTA-Tb3+@AFt-Rh-NO did not show observable responses to the additions of various ROS/RNS including ·OH, H2O2, 1O2, O2-, ClO-, ONOO-, NO2- and NO3-, while it was remarkably enhanced after PTTA-Tb3+@AFt-Rh-NO was reacted with NO. These results indicate that the luminescence response of the probe to NO is highly specific, which is attributed to the feature of Rh-NO in the probe, and provides high selectivity for the ratiometric time-gated luminescence detection of NO. The effect of pH on the I565/I539 ratio of PTTA-Tb3+@AFt-Rh-NO upon reaction with NO was investigated in 50 mM PBS buffers with different pH values ranging from 4.5 to 9.5. As shown in 12 ACS Paragon Plus Environment

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Figure 4, the I565/I539 ratios of PTTA-Tb3+@AFt-Rh-NO in the absence and presence of NO are not remarkably affected by the pH changes, which indicates that PTTA-Tb3+@AFt-Rh-NO can work well as a ratiometric time-gated luminescence probe for the detection of NO in weakly acidic, neutral, and weakly basic buffers. Luminescence Imaging of NO in Living Samples. The probe PTTA-Tb3+@AFt-Rh-NO is a protein-based probe with good biocompatibility and cell membrane permeability (apoferritin is one of the most widely used proteins in nature, and can be easily transferred into cells via an apoferritin receptor-mediated endocytosis process52), which enables it to be favorably used for the bioimaging. To evaluate its practical applicability for the ratiometric time-gated luminescence imaging of NO in living samples, the PTTA-Tb3+@AFt-Rh-NO-loaded HepG2 cells and Daphnia magna were prepared, and luminescence images of the samples in the absence and presence of NO were recorded both under steady-state and time-gated imaging modes. Figure 5 shows the bright-field, steady-state and time-gated luminescence images of the PTTA-Tb3+@AFt-Rh-NO-loaded HepG2 cells before and after reaction with NO (time-gated luminescence images were recorded with green filter, 540 ± 25 nm, for collecting Tb3+ luminescence signals and red filter, >590 nm, for collecting rhodamine luminescence signals, respectively). Before reaction with NO, green luminescence signals of the Tb3+ complex were obviously observed from the cells (Figure 5A-d), while red luminescence signals of rhodamine could hardly be observed (Figure 5A-c). However, after the cells were reacted with NO, both red luminescence signals (Figure 5B-c) and green luminescence signals (Figure 5B-d) were clearly observed from the cells. Furthermore, compared to the steady-state luminescence images (Figure 5A-b and Figure 5B-b), because the autofluorescence from the cells was completely suppressed by the time-gated imaging mode, highly specific and background-free time-gated luminescence images 13 ACS Paragon Plus Environment


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of the cells with clear red and green luminescence signals were obtained. In addition, when the PTTA-Tb3+@AFt-Rh-NO-loaded cells were co-incubated with NOC-13 and the NO scavenger c-PTIO,55 no any red luminescence signals of rhodamine could be observed (Figure S3 in Supporting Information), which demonstrates that the red time-gated luminescence signals from the cells in Figure 5B are truly attributed to the reaction of the probe with NO in the cells. These results reveal that the probe PTTA-Tb3+@AFt-Rh-NO could be easily transferred into living cells for probing the intracellular NO molecules to give the long-lived red and green luminescence signals, which demonstrated the applicability of the probe for the ratiometric time-gated luminescence imaging of NO in living cells. Daphnia magna, a widely used laboratory animal as an indicator of aquatic ecosystem health and as a model organism in ecotoxicology, were employed in this work to further confirm the applicability of PTTA-Tb3+@AFt-Rh-NO for imaging NO in living samples. As shown in Figure 6, after Daphnia magna were incubated with PTTA-Tb3+@AFt-Rh-NO for 1 h, only distinct Tb3+ green luminescence signals were observed (Figure 6A-d). When the Daphnia magna were further incubated with NO for another 20 min, red luminescence signals of rhodamine (Figure 6B-c) and green luminescence signals of Tb3+ complex (Figure 6B-d) were clearly observed. More importantly, compared to the steady-state luminescence images interfered by the strong autofluorescence, the time-gated luminescence images showed background-free images, which allowed the distributions of NO in the body of Daphnia magna to be clearly observed. This result demonstrated the applicable flexibility of PTTA-Tb3+@AFt-Rh-NO as a probe for the visualization of NO in small living animals.

CONCLUSIONS 14 ACS Paragon Plus Environment

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By assembling a luminescent Tb3+ complex and a NO-responsive rhodamine derivative into and onto apoferritin, a unique LRET-based rotiometric time-gated luminescence probe specific for NO, PTTA-Tb3+@AFt-Rh-NO, was successfully prepared in this work. In the presence of NO, the probe gives remarkably enhanced rhodamine emission and notably declined Tb3+ emission due to the existence of LRET process under a single-wavelength excitation (334 nm), which allows PTTA-Tb3+@AFt-Rh-NO to be used as a ratiometric probe for the recognition and time-gated luminescence detection of NO with the IRh/ITb ratio as a signal. Compared to the previously reported luminescence probes for NO, the new probe exhibits some distinct advantages, such as good water solubility, bioaffinity and biocompatibility, high selectivity and sensitivity, and ratiometric response ability with long-lived luminescence signals. These desirable features allow it to be favorably useful for the self-calibration ratiometric and background-free time-gated luminescence detection of NO in complicated biological samples. The results shown here of time-gated luminescence imaging to monitor the exogenous NO in living HepG2 cells and Daphnia magna demonstrated the applicability of the probe in vivo. These achievements demonstrated the utility of apoferritin as a modular and versatile scaffold for the preparation of a NO probe, which could be also a useful platform for the design of various ratiometric time-gated luminescence bioprobes, only with the replacement of Rh-NO by other bio-responsive rhodamine derivatives.

ASSOCIATED CONTENT Supporting Information. Preparation principle of PTTA-Tb3+@AFt-Rh-NO (Scheme S1), TEM image of the phosphotungstic acid-stained PTTA-Tb3+@AFt-Rh-NO (Figure S1), luminescence intensity decay curves of PTTA-Tb3+@AFt at 539 nm and PTTA-Tb3+@AFt-Rh-NO after reaction with NO at 565 nm (Figure S2), and luminescence images of the PTTA-Tb3+@AFt-Rh-NO-loaded 15 ACS Paragon Plus Environment


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HepG2 cells treated with NOC-13 and c-PTIO (Figure S3). This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENTS

Financial supports from the National Natural Science Foundation of China (Grant Nos. 21275025, 21477011), and the Specialized Research Fund for the Doctoral Program of Higher Education of China (Grant No. 20130041130003) are gratefully acknowledged.

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Figure Captions Scheme 1. Structures of PTTA-Tb3+ and Rh-NO (bottom), and general depiction of the luminescence response of PTTA-Tb3+@AFt-Rh-NO to NO (top). Scheme 2. Reaction pathway for the synthesis of Rh-NO. Figure 1. (A) Time-gated emission spectrum of PTTA-Tb3+ (λex = 334 nm, blue line) and the steady-state excitation (λem = 577 nm, black line) and emission (λex = 557 nm, pink line) spectra of 5-carboxytetramethylrhodamine. (B) Time-gated emission spectra (λex = 334 nm) of PTTA-Tb3+@AFt-Rh-NO before (blue line) and after (pink line) reaction with NO in 50 mM PBS buffer of pH 7.4. Figure 2. (A) Time-gated emission spectra (λex = 334 nm) of PTTA-Tb3+@AFt-Rh-NO (20 µL stock solution in 2.0 mL buffer) upon reaction with different concentrations of NO (0.0, 4.0, 10, 16, 20, 50, 100, 150, 200, 250, 300, 400, 500, 550, 600, 700, 800, 1000 µM) in 50 mM PBS buffer of pH 7.4 (the inset shows the variation of the I565/I539 ratio as a function of NO concentration). (B) Calibration curve for the ratiometric time-gated luminescence detection of NO. Figure 3. Effects of various ROS/RNS (1.0 mM) on the I565/I539 ratio of PTTA-Tb3+@AFt-Rh-NO (20 µL stock solution in 2.0 mL buffer) in 50 mM PBS buffer of pH 7.4. ROS/RNS: 1, blank; 2, NO (NOC-13 was used); 3, H2O2; 4, O2- (generated by xanthine+xanthine oxidase); 5, ·OH (generated by H2O2+ferrous ammonium sulfate); 6, 1O2 (generated by NaOCl+H2O2); 7, ClO-; 8, ONOO(3-morpholinosydnonimine, a ONOO- donor, was used); 9, NO2-; 10, NO3-. Figure 4. Effect of pH on the I565/I539 ratio variations of PTTA-Tb3+@AFt-Rh-NO before (●) and after (■) reaction with NO in 50 mM PBS buffers with different pH values. Figure 5. Bright-field (a), steady-state (b) and time-gated (c and d, recorded with red filter, >590 nm, for collecting rhodamine luminescence signals and green filter, 540 ± 25 nm, for collecting 20 ACS Paragon Plus Environment

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Tb3+

luminescence

signals,

respectively)

luminescence

images

of

the

PTTA-Tb3+@AFt-Rh-NO-loaded HepG2 cells before (A) and after (B) reaction with NO (NOC-13 was used). Scale bar: 10 µm. Figure 6. Bright-field (a), steady-state (b) and time-gated (c and d, recorded with red filter, >590 nm, for collecting rhodamine luminescence signals and green filter, 540 ± 25 nm, for collecting Tb3+

luminescence

signals,

respectively)

luminescence

images

of

the

PTTA-Tb3+@AFt-Rh-NO-loaded Daphnia magna before (A) and after (B) reaction with NO (NOC-13 was used). Scale bar: 200 µm.



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Excitation （ 334 nm）

Long-lived Tb3+ emission (λ λem=539 nm)

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Excitation （ 334 nm）

LRET Long-lived rhodamine emission (λ λem=577 nm)

NO PTTA-Tb3+

PTTA-Tb3+

PTTA-Tb3+@AFt-Rh-NO

N

N

N

O O

N N

= N

N Tb3+ O 2C CO2-

CO2- O2C PTTA-Tb

3+

N

N

O

O O

NH2

N

=

N

OH

= N

N

Rh-NO

Scheme 1


O Rhodamine

N

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O O

N

O

O

1

N

N O

O

N

O

DCC/NHS

ON

HN

N

O

DCC/DMAP

O-

O

O

N

COOH

N

O

O

H2N

NH2

O O

HCl TEA

N

NH2

N

NH N

O

N

H2N

2

N

O

3

Scheme 2


N

N

O

Rh-NO

N

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800

A 600

400

200

0 450

500

550

600

650

700

650

700

Wavelength (nm) Luminescence intensity (arb. unit)

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Luminescence intensity (arb. unit)


1000

B

800 600 400 200 0 450

500

550

600

Wavelength (nm)

Figure 1


1000

0.4

A I565/I539

0.3

800

0.1

565nm

600

0.2

539nm

0.0

0

200

400 600 800 Conc. of NO (µM)

1000

400 200 0 450

500

550

600

650

700

750

Wavelength (nm)

0.35

B

y = 0.0193 + 0.00104x, r = 0.994

0.30 0.25 I565/I539

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Luminescence intensity (arb. unit)

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0.20 0.15 0.10 0.05 0.00 0

100

200

300

400

500

Conc. of NO (µM)

Figure 2


600


0.35 0.30 0.25 I565/I539

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0.20 0.15 0.10 0.05 0.00 1

2

3

4

5

6

7

8

9

ROS/RNS

Figure 3


10

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0.4

0.3 I565/I539

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0.2

0.1

0.0 4

5

6

7

8

9

pH

Figure 4


10


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Figure 5


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Figure 6



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For TOC only

Excitation ）（ 334 nm）

Long-lived Tb3+ emission (λ λem=539 nm)

Excitation ）（ 334 nm）

LRET

NO PTTA-Tb3+

PTTA-Tb3+

PTTA-Tb3+@AFt-Rh-NO


Long-lived rhodamine emission (λ λem=577 nm)

Ratiometric Time-Gated Luminescence Probe for Nitric Oxide Based

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