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NIR ratiometric luminescence detection of pH fluctuation in living cells with hemicyanine derivative-assembled upconversion nanophosphors Haixia Li, Hao Dong, Ming-Ming Yu, Chunxia Liu, ZhanXian Li, Liu-He Wei, Lingdong Sun, and Hongyan Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b01324 • Publication Date (Web): 14 Jul 2017 Downloaded from http://pubs.acs.org on July 14, 2017

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Analytical Chemistry

NIR ratiometric luminescence detection of pH fluctuation in living cells with hemicyanine derivative-assembled upconversion nanophosphors Haixia Li,†,∥ Hao Dong,‡,∥ Mingming Yu,† Chunxia Liu,† Zhanxian Li,*,† Liuhe Wei,† Ling-Dong Sun,*,‡ and Hongyan Zhang*,§ †

College of Chemistry and Molecular Engineering, Zhengzhou University, Zhengzhou, Henan

450001, China. *Tel: +86 0371 67783126. E-mail: [email protected]. ‡

Beijing National Laboratory for Molecular Sciences, State Key Lab of Rare Earth Materials

Chemistry and Applications, PKU-HKU Joint Laboratory in Rare Earth Materials and Bioinorganic Chemistry, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China. *E-mail: [email protected]. §

Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical

Institute of Physics and Chemistry, Chinese Academy of Sciences, Beijing, 100190, China. *Fax: +86 10 82543435. Tel.: +86 10 82543435. E-mail: [email protected].

S1 ACS Paragon Plus Environment


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ABSTRACT It is crucial for cell physiology to keep the homeostasis of pH, and it is highly demanded yet challenging to develop luminescence resonance energy transfer (LRET) based near infrared (NIR) ratiometric luminescent sensor for the detection of pH fluctuation with NIR excitation. As promising energy donors for LRET, upconversion nanoparticles (UCNPs) have been widely used to fabricate nanosensors, but the relatively low LRET efficiency limits their application in bioassay. To improve the LRET efficiency, core/shell/shell structured βNaGdF4@NaYF4:Yb,Tm@NaYF4 UCNPs were prepared and decorated with hemicyanine dyes as an LRET-based NIR ratiometric luminescent pH fluctuation-nanosensor for the first time. The as-developed nanosensor not only exhibits good anti-disturbance ability, but also can reversibly sense pH, and linearly sense pH in a range of 6.0−9.0 and 6.8−9.0 from absorption and upconversion emission spectra respectively. In addition, the nanosensor displays low dark toxicity under physiological temperature, indicating good biocompatibility. Furthermore, live cell imaging results revealed that the sensor can selectively monitor pH fluctuation via ratiometric upconversion luminescence behavior.


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As an important physiological parameter, pH plays an important role in biomedical research.1 Aberrant fluctuation of pH symbolizes dysfunctions of cells and diseases.2,3 Moreover, many biological processes are affected with basic pH. For instance, mitochondria displays a slightly basic pH,4–6 and plays significant key roles in a great deal of cellular biological processes.7–16 The physiological pHi deviation is minor yet very significant for the understanding of physiology. Hence, it is highly needed to develop a novel sensor which can reliablely, sensitively and accurately monitor pHi in living cells. Fluorescent detection has become an indispensable tool to investigate biological events because of its simple operation, fast response, high selectivity, high sensitivity, and unparalleled

spatiotemporal

resolution.

Fluorescence

imaging

has

become

an

indispensable tool to investigate biological events because of its simple operation, fast response, and spatial-temporal resolution.17–19 Up to now, a lot of pH sensors including organic molecules20,21 and nanophosphors22–29 have been developed and most of them are off-on sensors toward acidic pH. Using the ratio of two different wavelengths emission intensities to realize ratiometric imaging is considered as a more rational approach to monitoring pHi deviation, because the emission intensity can be influenced by a lot of factors.30–32 Luminescence resonance energy transfer (LRET) has been widely adopted in ratiometric detection because of its facile control and basis on established theory.33–36 Untill now, the reported ratiometric pHi sensors especially basic pHi sensors are still limited, and it is crucial to design LRETbased ratiometric luminescent sensors for basic pHi.



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In addition, most of the reported pH sensors have one general problem when they are used in biological systems: they have very short excitation and emission wavelengths, and the few available sensors absorbing in the biological optical window in the far red or NIR spectral range are prone to photobleaching.37–43 Upconversion luminescence (UCL) as the detection signal has many advantages, such as a large anti-Stokes shift, an absence of autofluorescence from biological samples, remarkable light penetration depth in tissue due to its NIR excitation, and no photobleaching,44–46 which offers us a promising strategy for the design of luminescent nanosensors for pH. Lanthanide-doped upconversion nanoparticles, such as NaYF4:Yb,Er and NaYF4:Yb,Tm show high upconversion efficiency upon 980 nm excitation, which have been applied as LRET donors for sensing metal ions, anions and biological molecules with small-molecule organic dyes as LRET acceptors.47–49 However, very few examples of upconversion nanocrystal-small molecular hybrid sensors toward pH have been reported.50–54 Therefore, exploration of upconversion nanocrystal-small molecular nanocomposites as ratiometric luminescent sensors with high LRET efficiency to monitor and image pH deviation in living cells or biological processes is still a great challenge. Since the LRET efficiency depends closely on the spatial distance between energy donor and energy acceptor, it is important to rationally design the upconversion nanoparticles for improving the LRET efficiency. In this paper, to shorten the distance between

upconversion

upconversion

luminescent

nanoparticle

was

centers

designed

and as

organic

small

core/shell/shell

molecules, structure

of

the β-

NaGdF4@NaYF4:Yb,Tm@NaYF4 (UCNPs). Yb and Tm are doped in the intermediate shell layer, which made them distributed in the more outer space and are closer to the


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energy acceptor. The outmost NaYF4 shell acts as the protecting layer to enhance the overall emission efficiency.53,55 As a result, it is easier to activate the LRET process from UCNPs to organic molecules. Using assembling method, the first example of an LRETbased ratiometric pHi-upconversion nanosensor was described. In our sensor, the UCNPs serve as energy donor and anchoring site. The schematic illustration for the sensing of pHi by the UCNPs-based sensor was shown in Scheme 1. Our sensor demonstrates some advantages: firstly, the excitation wavelength was 980 nm, which is suitable for measurements in complex biological samples; secondly, it is a ratiometric sensor which can more accurately detect minor pHi deviation in biosystem; thirdly, it can reversibly sense pH and linearly response pH in certain pH range; lastly, with this sensor, ratiometric pHi imaging has also been successfully demonstrated via confocal microscopy. EXPERIMENTAL SECTION Methods and Materials. p-Hydrazinobenzoic acid hydrochloride, Dimethylacetone, CH3I, AcOH, AcONa, 4-hydroxybenzaldehyde, Cys (cysteine), Glu (glutamate), GSH (glutathione), H2O2, Pro (proline), Asp (aspartic acid), Ala (alanine), Arg (arginine), lle (isoleucine), Glc (glucose), Lys (lysine), Phe (phenylalanine), Tyr (tyrosine), Met (methionine), Leu (leucine), Hcy (hemocyanin), Ser (serine), Gly (glycine), Trp (tryptophan), Val (valine), Thr (threonine), AlCl3, CaCl2, CdCl2, CoCl2, CuCl2, HgCl2, KCl, MgCl2, MnCl2, NaCl, NiCl2, Pb(NO3)2, ZnCl2, NaF, NaBr, NaI, Na2S, Na3N, NaHS, Na2CO3, NaNO3, Na2SO3, Na2SO4, Na2SiO3, Na3PO4, NaClO3, NaClO4, Na2C2O4, Na2S2O3, Na4P2O7, NaHCO3, NaHSO3, Na2HPO4 and NaH2PO4, trifluoroacetic acid (99%), 1-octadecene (ODE, 90%), oleic acid (OA, 90%), polyacrylic acid (PAA, Mw = 1,800), dimethylformamide (DMF), nitrosonium tetrafluoroborate (NOBF4, 98%)



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were used directly without purification. Water used in all experiments was obtained using a Milli-Q water system. 1

H NMR (400 MHz) and 13C NMR (100 MHz) spectra were obtained on a Bruker Advance-

400 spectrometer, with tetramethylsilane as an internal standard. The HRMS spectra were measured with Fourier-transform ion cyclotron resonance mass spectrometry (IonSpec4.7 Tesla FTMS-MALDI/DHB). All absorption and UCL spectra in this work were obtained with a TU1901 and a Hitachi F4600 fluorescence spectrometer with high stability of 980 nm pump laser source at room temperature, erspectively. Samples for transmission electron microscopy (TEM) analysis were prepared by drying a colloidal solution of nanoparticles on amorphous carboncoated copper grids. Low-resolution TEM were operated on a JEOL-JEM 2100 transmission electron microscopy operated at 200 kV. High resolution TEM (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) were carried out on a JEOL-JEM 2100F field emission transmission electron microscope operated at 200 kV. Powder X-ray diffraction (XRD) patterns were recorded on a Rigaku D/MAX-2000 diffractometer (Japan) with a slit of 1/2° at a scanning rate of 2 °C/min, using Cu Kα radiation ( = 1.5406 Å). Upconversion emission spectra were recorded on a Hitachi F-4600 spectrometer, with the PMT voltage of 700 V. Cell incubation and imaging. HeLa cells were seeded in a 12-well plate in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum and 1% penicillin. The cells were incubated under an atmosphere of 5% CO2 and 95% air at 37 °C for 24 h before the cell imaging experiments. The cells were washed three times with PBS buffer before used. Luminescence imaging experiments in Living Hela cells were operated with Nikon


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A1Rmp-PicoQuant FLIM. 980 nm semiconductor laser was used for luminescence imaging experiments. Cell confocal experiments under different pH conditions. HeLa cells were cultured on the culture dishes, then 20 μL 1 mM Nigeria, 100 μL 2.4 M KCl, 880 μL 1640 culture medium, and 1 mL pH 6.0/7.0/8.0/9.0 buffer were added, which were incubated with HeLa cells for 30 min. PAA-UCNPs-1 was added to the different pH cell suspensions and the concentration of the nanosensor was kepet as 0.15 mg/mL. The nanosensor and the cells were incubated for 20 min. Synthesis of sensor 1. The mixture of compound 2 (Scheme S1) (345 mg, 1.0 mmol), 4hydroxybenzaldehyde (122 mg, 1.0 mmol), and 5 mL ethanol was refluxed for 3 h. The mixture was cooled to room temperature. After filtration and washing the filter cake with ethanol, compound 1 (Schemes 1 and S1) (159.3 mg, 31.6%) was obtained.52 Characterization of 1: 1H NMR (400 MHz, DMSO-d6, δ): δH 10.97(s, 1H), 8.47 (d, 1H), 8.39 (s, 1H), 8.17 (m, 3H), 7.93 (d, 1H), 7.49 (d, 1H), 6.98 (d, 2H), 4.10 (s, 3H), 1.82 (s, 6H). 13C NMR (100 MHz, DMSO-d6, δ): δC 183.80, 167.08, 164.30, 155.91, 145.69, 143.86, 134.60, 131.16, 130.87, 126.53, 124.20, 117.03, 115.13, 109.76, 52.26, 34.69, and 26.06. HRMS-ESI m/z: calcd for C20H20NO3 [M-I], 322.1443; found, 322.1442. Synthesis of α-NaGdF4 nanoparticles. Gd(CF3COO)3 (1 mmol) and CF3COONa (1 mmol) were added to a mixture containing oleic acid (OA), oleylamine (OM), and octadecene (ODE) (40 mmol, molar ratio: 1:1:2) into a three-necked flask (100 mL) at room temperature. The slurry was heated to 110 °C to remove water and oxygen with vigorous stirring under vacuum. Then, the solution was heated to 310 °C and kept for 15 min under N2 atmosphere. After cooling to room temperature, the nanoparticles were collected with centrifugation (7800 rpm, 10 min) after



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precipitation by adding excess amount of ethanol. The final product was dispersed in 10 mL cyclohexane. Synthesis of β-NaGdF4 nanoparticles. 5 mL as-prepared α-NaGdF4 nanoparticles were dispersed in a mixture of OA and ODE (40 mmol, molar ratio: 1:1). After that, 0.5 mmol Gd(CF3COO)3 and 0.5 mmol CF3COONa were added. After removing cyclohexane, water, and oxygen with vigorous stirring under vacuum, the mixture was then heated to 310 °C and kept for 30 min under N2 atmosphere. Then, the reaction was cooled to room temperature. Excess amount of ethanol was added to precipitate the nanoparticles, which can be collected by further centrifugation (7800 rpm, 10 min). The resulting nanoparticles were redispersed in 10 mL cyclohexane. Synthesis of β-NaGdF4@NaYF4:Yb,Tm nanoparticles. 5 mL as-prepared β-NaGdF4 colloidal solutions were dispersed in a mixture of OA and ODE (40 mmol, molar ratio: 1:1). Then, a given amount of Ln(CF3COO)3 (0.09 mmol), Y(CF3COO)3 (0.09 mmol), 0.9 mmol Yb(CF3COO)3, 0.01 mmol Tm(CF3COO)3) and CF3COONa (1 mmol) were added. After degassing, the solution was heated to 310 °C and kept for 30 min under N2 atmosphere. Then, the system was cooled to room temperature, and the nanoparticles were collected with centrifugation (7800 rpm, 10 min) after precipitation by adding excess amount of ethanol. The final product was dispersed in 10 mL cyclohexane. Synthesis of β-NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles. 5 mL colloidal solution containing as-prepared β-NaGdF4@NaYF4:Yb,Tm nanoparticles were dispersed in a mixture of OA and ODE (40 mmol, molar ratio: 1:1). Then, 1 mmol Y(CF3COO)3 and 1 mmol CF3COONa were added. After degassing, the solution was heated to 310 °C and kept for 30 min under N2


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atmosphere. After cooling to room temperature, excess amount of ethanol was added to precipitate the nanoparticles, which can be collected by further centrifugation (7800 rpm, 10 min). The final product was dispersed in 5 mL cyclohexane. Preparation of PAA modified upconversion nanoparticles. 1 mL cyclohexane colloidal solution containing hydrophobic β-NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles was dispersed in a mixture of 5 mL N,N-Dimethylformamide (DMF) and 4 mL cyclohexane. Then, 50 mg NOBF4 was added. After stirring for 30 min, the DMF phase was taken out to be precipitated with excess amount of tolune (18000 rpm, 15 min). The products were redispersed into 10 mL DMF containing 30 mg PAA (25% saponification). Then, the system was stirred overnight. Finally, PAA modified nanoparticles were collected after adding excess amount of acetone and centrifugation (18000 rpm, 15 min). Assembly of sensor 1 on the Surface of PAA-UCNPs (PAA-UCNPs-1). DMSO (0.5 mL) solution of sensor 1 (0.03 mmol) was added dropwise into a water solution of the prepared PAAUCNPs (1 mg/mL) in a round-bottomed flask, and then the mixture was stirred overnight at room temperature to obtain a homogeneous phase. Then, the mixture was centrifuged, and the collected solid was repeatedly washed with water/ethanol (v/v = 1:1) by centrifugation. The precipitate could be dispersed in deionized water or other polar solvents. RESULTS AND DISCUSSION Synthesis of PAA-UCNPs, and PAA-UCNPs-1 The β-NaGdF4@NaGdF4:Yb,Tm@NaYF4 core/shell/shell nanoparticles were prepared with a modified thermal decomposition method, while the PAA modified UCNPs (PAA-UCNPs) with a



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ligand exchange process.53–57 The TEM images of β-NaGdF4, β-NaGdF4@NaYF4:Yb,Tm, and β-NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles (Figures 1a, 1b and 1c) reveal that the nanoparticles are uniform in size and morphology, and have a diameter ranging from 13 to 30 nm. The typical HRTEM image of as-prepared β NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles exhibit a legible lattice distance of 0.52 nm, corresponding to the (100) plane of βNaREF4 (Figure 1d). This suggests the formation of hexagonal phased structure. Figure S1 demonstrates the XRD patterns of as-prepared β-NaGdF4 (core), β-NaGdF4@NaYF4:Yb,Tm (core@shell), and β-NaGdF4@NaYF4:Yb,Tm@NaYF4 (core@shell@shell) nanoparticles, which are consistent with the standard pattern referenced below (JCPDS 16-0334). The core/shell/shell structure can be clearly noticed through image contrast in the HAADF-STEM image (Figure 1e). The inner bright region corresponds to lanthanide elements in NaGdF4@NaYF4:Yb,Tm, while the outer dark region relates to Y element in NaYF4 shell (Figure 1e). Figure 1f shows that there is no obvious change in size and morphology of nanoparticles after ligand exchange, and the nanoparticles are dispersed well in aqueous phase. Figure S2 depicts the typical upconversion emission spectra of as-prepared UCNPs, from which we can find the typical UV, visible, and NIR upconversion emissions of Tm3+. The blue line in Figure S2 indicates that the upconversion emission from PAA modified UCNPs drops a little due to the quenching from water molecules and the absolute quantum yield was 6.5%.58 In the FTIR spectrum of PAA-UCNPs-1 (see Figure S3), the peaks at 1473, 1527, and 1573 cm-1 can be assigned to sensor 1, indicating the successful assembly of 1 with PAA-UCNPs.


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Scheme 1. Schematic illustration (not to real scale) of the LRET-based ratiometric nanosensor toward pH from hemicyanine dyes modified core/shell/shell UCNPs.



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Figure

1.

TEM

images

of

β-NaGdF4

(a),

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β-NaGdF4@NaYF4:Yb,Tm

(b),

β-

NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles (c). (d) Typical HRTEM image of as-prepared β-NaGdF4@NaYF4:Yb,Tm@NaYF4

nanoparticles.

(e)

HAADF-STEM

image

of

β-

NaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles. (f) TEM image of PAA modified βNaGdF4@NaYF4:Yb,Tm@NaYF4 nanoparticles. (g) Absorption spectra of sensor 1 (1.0 × 10−5


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M) in 0.01 M different pH HEPES solution (green, red, and blue lines) and luminescence spectra of PAA-UCNPs (0.15 mg/mL) with excitation at 980 nm (black line) in pH 7.4 HEPES solution. The absorption peak at 535 nm in the UV-vis absorption spectrum of PAA-UCNPs-1 dispersed in DMSO–HEPES buffer solution (1:9, v/v) was used to determine the amount of sensor 1 loaded in the nanosystem (see Figure S4). As shown in the inset of Figure S4, the absorption intensity at 535 nm is 0.489. According to a standard curve plotted by examining free sensor 1 solutions at different specified concentrations, the amount of sensor 1 in the hybrid material was estimated to be ~7.07 wt%. pH sensing properties of PAA-UCNPs and compound 1 As shown in Figure 1g, the maximum absorption peaks of compound 1 at pH of 5.0 and 9.0 are at about 432 and 535 nm respectively and the emission peaks of PAA-UCNPs are at about 450, 475, and 513 nm in pH range from 4.0 to 9.0 upon excitation at 980 nm. In acidic pH, the emission of PAA-UCNPs at 450 and 475 nm can be absorbed by compound 1. While in basic pH, the emission of PAA-UCNPs at 513 nm can be absorbed by compound 1, indicating the possibility of LRET from Yb3+–Tm3+ pairs to compound 1. (Schemes 1 and S1). Therefore, it is possible to select a 980 nm wavelength laser to excite PAA-UCNPs in the hybrid sensor. Upon excitation at 980 nm and in acidic pH, the emission of PAA-UCNPs at 450 and 475 nm was absorbed by compound 1 and the 513 nm emission of the nanophosphor doesn't change. While in neutral and basic pH, the emission at 475 and 513 nm of PAA-UCNPs was absorbed by compound 1 (Schemes 1 and S1) and no change in the 450 nm emission was observed. In addition, whenever in acidic, neutral or basic pH, the emission at 655 nm of PAA-UCNPs cannot be absorbed by compound 1.



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Figure S5 and Figure S6 present the fluorescence spectra of compound 1 and upconversion emission spectra of PAA-UCNPs in different pH conditions. It is clear that the emission of 1 (Scheme S1) (1.0 × 10−5 M HEPES solution) at 525 nm decreased and an emission peak at 565 nm increased dramatically with pH increasing (Figure S5). The high pH- induced formation of compound 4 (Schemes 1 and S1) from sensor 1 is responsible for the absorption and fluorescence spectral change of sensor 1.59,60 However, the emission spectrum of the PAAUCNPs (0.15 mg/mL HEPES solution) hardly changes upon pH changing from 4.0 to 9.0 (Figure S6). Such results indicate that pH change can result in the fluorescence change of 1 and have no influence on the luminescence spectrum of PAA-UCNPs. Prior to sensor preparation, the interaction between PAA-UCNPs and sensor 1 was investigated by monitoring their absorption and fluorescence spectral changes.


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Figure 2. (a) Upconversion emission spectra of PAA-UCNPs-1 (0.15 mg/mL) in DMSO– HEPES buffer solutions (1:9, v/v) of pHapp values under excitation at 980 nm. Inset: amplified emission spectra of PAA-UCNPs-1 from 500 to 550 nm. (b) The pH titration curve was plotted by the ratiometric emission spectra of PAA-UCNPs-1 as linear function of pHapp value. All the RSD values were less than 5.0% (Mean of three replicates). pH sensing properties of PAA-UCNPs-1 The luminescence spectra of PAA-UCNPs-1 at different apparent pH (pHapp) were examined in a HEPES buffer containing 10% DMSO. As shown in Figure S7 in Supporting Information, when pHapp increased from 4.0 to 9.0, an intense band at 430 nm of PAA-UCNPs-1 decreased and a new peak at 535 nm increased. Concomitantly, the luminescence at 513 nm decreased and that at



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650 nm hardly changed in intensity with increased pHapp (Figure 2(a)). The changes in absorption and upconversion emission spectra are ascribed to the formation of compound 4 (Schemes 1 and S1) and the subsequent LRET from energy donor PAA-UCNPs to energy acceptor sensor 1. Via the pH titration function of UCL650/UCL513 value (see Figure S8), the apparent pKa' was confirmed to be 8.05 ± 0.36.61 The linear range for the response from absorption and upconversion emission spectra of PAA-UCNPs-1 are pHapp of 6.8 to 8.4 (Figure S9) and 6.8 to 9.0 (Figure 2(b)) respectively. In addition, the enhanced UCL650/UCL513 value of PAA-UCNPs-1 at pHapp 9.0 recovered to the solution original UCL650/UCL513 value at pHapp 6.6, UCL650/UCL513 value shows high stability in seven cycles for each pH, demonstrating that PAAUCNPs-1 owns excellent reversibility in the pHapp range (Figure S10(a)). Compared with the reported sensors toward pH fluctuation (Table S1 in Supporting Information), our present work indicates some advantages such as excitation at 980 nm and NIR ratiometric fluorescent detection mode, which make this sensor more appropriate to be applied in sensing intracellular pH. As shown in Figure S10(b), when temperature increases from 30 to 48°C, the UCL ratio UCL650/UCL513 of 0.15 mg/mL PAA-UCNPs-1 in DMSO–HEPES buffer solutions (1:9, v/v, pHapp = 7.4) changes very little, demonstrating that PAA-UCNPs-1 is a thermal stable sensor. Photostability experiment showed that UCL650/UCL513 doesn't change with time extending (Figure S11). The results demonstrate that PAA-UCNPs-1 has excellent thermal stability and photostability. Specific response to pH


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The UCL emission intensity of PAA-UCNPs-1 at pH = 7.4 showed little changes upon addition of common species including metal ions, anions, and biological molecules (see figures 12, S13 and S14) was also tested. The results demonstrated that all of the selected species have no interference in the sensing of pHapp except Cu2+. The interference from Cu2+ may be ascribed to the coordination interaction between phenolic hydroxyl group and Cu2+. This result strongly indicates that PAA-UCNPs-1 could be a good ratiometric UCL sensor towards pH with strong anti-interference ability. Reproducibility To determine the reproducibility of the present sensor system, each sample was measured in three independent experiments, which are shown in figures 2, S8, S9, S10, S11, S12, S13, S14, and S15. All the RSD values were less than 5.0% (Mean of three replicates). The reproducibility results obtained from the thermal stability of emission intensity ratio of I650/I513 are shown in Table S2. Imaging of PAA-UCNPs-1 to pH in live cells Standard MTT assays were performed to evaluate the cytotoxicity of the sensor PAA-UCNPs-1 in live cells. When the concentration of PAA-UCNPs-1 reached 13.6 μg/mL for 1 h, the cell viabilities were still higher than 80%, suggesting the low cytotoxicity of PAA-UCNPs-1 (see Figure S15). The practical ratiometric pHi imaging experiment of PAA-UCNPs-1 was carried out in monitoring pHi fluctuation (Figure 3 and Figure S16). When the concentration of PAAUCNPs-1 was 0.15 mg/mL, the intensities of green (Figures 3a, 3f, 3k and 3p) and red (Figures 3b, 3g, 3i and 3q) luminescence were increased with pH increasing, such results may be ascribed to the more nanocomposites which entered the cells. Fortunately, the ratio of Igreen to Ired



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gradually increased with pH changing from 6.0 to 9.0, indicating the advantage of the ratiometric fluorescence imaging. The change in the ratio of Igreen to Ired shows the ability of sensor PAAUCNPs-1 towards a pH-dependent signal. The ratio value of Igreen/Ired is 7.211, 6.091, 2.059 and 1.973 from pH 6 to pH 9.

Figure 3. Confocal microscopy images of HeLa cells incubated with PAA-UCNPs-1 (0.15 mg/mL) at different pH (6, a–d), (7, f–i), (8, k–n), and (9, p–s). The excitation wavelength was 980 nm. The images were collected at green channel with 525/50 filter (a, f, k, p), red channel with 595/50 filter (b, g, l, q), overlay (c, h, m, r), and Ratio images (d, i, n, s). The ratio value of Igreen/Ired is 7.211, 6.091, 2.059 and 1.973 from pH 6 to pH 9. CONCLUSION In summary, a novel nanoplatform of pH-insensitive PAA modified core/shell/shell βNaGdF4@NaYF4:Yb,Tm@NaYF4 UCNPs and a pH fluctuation sensitive hemicyanine derivative led to the first LRET-based NIR ratiometric luminescent pH fluctuation nanosensor. The excitation of 980 nm light, UCNPs exhibits the LRET-based NIR ratiometric luminescent response of pH under physiological temperature. NIR ratiometric sensor PAA-UCNPs-1 has


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been successfully used for pHi detection in live cells. This work provides not only an imaging nanomaterial for pHi but also an example of a composite method to construct NIR ratiometric pHi sensors. The intrinsic limitations of sensor PAA-UCNPs-1 focus on its too few amount which enter the living cells in acidic condition, which may limit its application. With the rapid development in organic and inorganic synthesis methods, this disadvantage could be overcome in the near future. ASSOCIATED CONTENT Supporting Information. Text and figures giving additional experimental details, including spectral data, images of the chromatography plates, and crystallographic data. This material is available free of charge via the Internet at http://pubs. acs.org. Experimental details of synthesis route to sensor 1, its sensing mechanism and XRD patterns of

as-prepared

β-NaGdF4

(core),

β-NaGdF4@NaYF4:Yb,Tm

(core@shell),

and

β-

NaGdF4@NaYF4:Yb,Tm@NaYF4 (core@shell@shell) nanoparticles, FT-IR of sensor 1, PAAUCNPs-1 and PAA-UCNPs, and upconversion spectra of compound 1, oleic acid capped UCNPs (OA-UCNPs), and PAA modified UCNPs (citric-UCNPs), and the anti-disturbance experiments of PAA-UCNPs-1 for pH detection, and additional cell experiments of sensor PAAUCNPs-1, table of comparison of various fluorescent sensors employed for pH fluctuation sensing and reproducibility of sensor PAA-UCNPs-1. AUTHOR INFORMATION Corresponding Author



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*Fax:

86

371-67781205.

E-mail

addresses:

[email protected],

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[email protected],

or

[email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This study was supported by Key Lab. of Photochem. Conver. Optoelect. Mater., Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, NNSF of China (21601158, U1504203, and 21425101), and Zhengzhou University (32210431). REFERENCES (1) Andriole, G. L.; Grubb, R. L., III; Buys, S. S.; Chia, D.; Church, T. R.; Fouad, M. N.; Gelmann, E. P.; Kvale, P. A.; Reding, D. J.; Weissfeld, J. L.; Yokochi, L. A.; Crawford, E. D.; O'Brien, B.; Clapp, J. D.; Rathmell, J. M.; Riley, T. L.; Hayes, R. B.; Kramer, B. S.; Izmirlian, G.; Miller, A. B.; Pinsky, P. F.; Prorok, P. C.; Gohagan, J. K.; Berg, C. D.; Team, P. P., New Engl. J. Med. 2009, 360, 1310–1319. (2) Brenner, C.; Galluzzi, L.; Kepp, O.; Kroemer, G., J. Hepatol. 2013, 59, 583–594. (3) Wiig, H.; Swartz, M. A., Physiol. Rev. 2012, 92, 1005–1060. (4) Casey, J. R.; Grinstein, S.; Orlowski, J., Nat. Rev. Mol. Cell Bio. 2010, 11, 50–61. (5) Leung, C. W. T.; Hong, Y. N.; Chen, S. J.; Zhao, E. G.; Lam, J. W. Y.; Tang, B. Z., J. Am. Chem. Soc. 2013, 135, 62–65. (6) Tantama, M.; Hung, Y. P.; Yellen, G., J. Am. Chem. Soc. 2011, 133, 10034–10037.


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NIR Ratiometric Luminescence Detection of pH ... - ACS Publications

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