Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection

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Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection and Imaging of Ascorbic Acid in Living Cells and Tissues Hong-Min Meng, Xiao-Bing Zhang, Chan Yang, Hai-lan Kuai, Guojiang Mao, Liang Gong, Wenhan Zhang, Suling Feng, and Junbiao Chang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01352 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 12, 2016

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Analytical Chemistry

Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection and Imaging of Ascorbic Acid in Living Cells and Tissues †

‡

‡

‡

†

‡

Hong-Min Meng , Xiao-Bing Zhang*, , Chan Yang , Hailan Kuai , Guo-Jiang Mao , Liang Gong , ‡

†

Wenhan Zhang , Suling Feng and Junbiao Chang*,

†

† Collaborative Innovation Center of Henan Province for Green Manufacturing of Fine Chemicals,Key Laboratory of Green Chemical Media and Reactions, Ministry of Education, School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, Henan 453007, P. R. China

‡ Molecular Sciences and Biomedicine Laboratory, State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China * To whom correspondence should be addressed. [email protected]；[email protected]

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ABSTRACT: Ascorbic acid (AA) serves as a key coenzyme in many metabolic pathways, and its abnormal level is found to be associated with several diseases. Therefore, monitoring AA level in living systems is of great biomedical significance. In comparison with one-photon excited fluorescent probes, twophoton (TP) excited probes are more suitable for bio-imaging, as they could afford higher imaging resolution with deeper imaging depth. Here, we report for the first time an efficient TP fluorescence probe for turn-on detection and imaging of AA in living cells and tissues. In this nanosystem, the negatively charged two-photon nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with two-photon dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge by electrostatic force, leading to remarkable decrease in their fluorescence intensity. However, the introduction of AA could induce the fluorescence recovery of the nanoprobe. Since it could reduce CoOOH into Co2+, and resulting in the destruction of the CoOOH nanoflakes. The nanosystem exhibits a high sensitivity towards AA, with a LOD of 170 nM observed. It also shows high selectivity toward AA over common potential interfering species. The nanoprobe possessed both the advantages of TP imaging and excellent membrane-permeability and good biocompatibility of the silica nanoparticles, and was successfully applied in TP-excited imaging of AA in living cells and tissues.


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INTRODUCTION Monitoring the distribution of reactive species in living systems is of great importance for understanding their physiological functions and pathological effects, as well as early-stage diagnosis of some serious diseases. For example, ascorbic acid (vitamin C, AA), which is an important reactive biological molecule in the human body, acts as many roles including enzyme cofactor, antioxidant against oxidative damages and involvement in neurotransmitter-related enzymes.1,2 In addition, a variety of epidemiologic studies and clinical trials have shown that the abnormal level of AA is associated with many diseases, such as scurvy, depression, connective tissue defects and diarrhea.3,4 Therefore, the development of efficient methods for monitoring AA level in living systems has become an important subject of current chemical research. Up to now, several methods including chromatography,5 electrochemical analysis,6,7 and fluorescence assay

8,9

have been developed for the detection of AA in vitro or in vivo. For the

bioimaging of AA in living systems, fluorescent probe-based imaging technique might be an ideal candidate due to its intrinsic advantages such as high sensitivity, real-time and in-situ monitoring and non-sample damaging.10-13 So far, several organic molecule-based fluorescent probes have been reported for AA assay.14,15 However, there are several obstacles, such as, photobleaching, poor membranepermeability and water-solubility, which strictly limit their biological applications. Various nanomaterials, such as quantum dots, MnO2 nanosheets, and carbon nanomaterials, which possess good membrane-permeability and excellent biocompatibility, have also been employed for designing of fluorescent probes for sensing AA in the past decade. Yan group developed a CdTe QDbased fluorescent nanoprobe for selectively detecting AA in biological fluids.16 By absorbing 7hydroxycoumarin on single-layer MnO2 nanosheets, Mao group reported a new fluorescent nanoprobe for in vivo sensing of AA in rat brain.17 Based on the specific oxidation-reduction reaction between CoOOH nanoflakes and AA, Lin et al. reported CoOOH nanoflakes-modified carbon dots-based fluorescent nanoprobe for monitoring cerebral AA in brain microdialysate.18 Although they are useful, 3 Environment ACS Paragon Plus


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these probes are based on traditional one-photon excited technology with short excitation wavelengths, which suffered from photobleaching, interference from bio-related autofluorescence, and limited penetration depth. Therefore, for monitoring AA in complex biosystems, it is desirable to develop efficient nanoprobes with low fluorescent background and deep penetration depth. To address this issue, Tang group reported a CoOOH nanoflake-modified persistent luminescence nanoparticle as nanoprobe for detection and imaging of AA in living cells and in vivo, which permits detection and imaging of AA without external excitation, and is free from bio-related autofluorescence interference. 19 Two-photon fluorescent microscopy (TPM), which employed near-infrared laser pulses for excitation, afforded a number of advantages over traditional one-photon microscopy, including less fluorescence background, light scattering, deeper penetration depth, and less tissue injury with prolonged observation time. Moreover, compared with persistent luminescence, TPM possessed higher imaging resolution, better three-dimensional spatial localization and deeper penetration depth.

20-22

These advantages make TP fluorescence probe more suitable for bio-imaging applications. Recently, many TP probes based on organic fluorophores or nanomaterials have been developed for detection and bioimaging of metal ions, anions and neutral molecules.23-31 Among them, nanoprobes are much more feasible for intracellular imaging owing to their nano-endocytosis effect induced good membranepermeability. To our best knowledge, no two-photon probe has been reported for AA detection up to date. In this work, by combining two-photon nanoparticles (TPNPs) with CoOOH nanoflakes, we try to develop a two-photon fluorescence nanoprobe (CoOOH-Modified TPNPs) for monitoring of AA in living systems. In this nanoprobe, TPNPs served as fluorescence reporter, while CoOOH nanoflakes acted as both fluorescence quenchers and AA recognition units (Scheme 1). The negatively charged two-photon nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with two-photon dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge through electrostatic force. Since the emission spectrum of TPNPs overlaps well with the absorption spectrum of 4 Environment ACS Paragon Plus

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CoOOH nanoflakes, the fluorescence of TPNPs can be efficiently quenched by the nanoflakes via energy transfer (ET). In the presence of AA, the CoOOH nanoflakes on the surface of TPNPs are reduced into Co2+, and the fluorescence of TPNPs is remarkably restored due to the decomposion of the CoOOH nanoflakes. The two-photon nanoprobe exhibited high sensitivity to AA, with a limit of detection (LOD) of 170 nM achieved. The nanoprobe also shows high selectivity to AA over common potential interfering species, as well as excellent cellular membrane-permeability and good biocompatibility. At last, the nanosystem is applied for TP excited fluorescence imaging of AA in living cells and tissues with satisfactory result.

Scheme 1. Preparation of CoOOH-Modified TPNPs (A) and Detection and Two-photon imaging of Ascorbic Acid in Living Cells and Tissues through CoOOH-Modified TPNPs (B). EXPERIMENTAL SECTION Reagents.

Unless

specified,

Hexadecyltrimethylammonium

reagents

bromide

were

(CTAB),

used

without

further

3-isocyanatopropyltriethoxysilane

purification. (3-ICPES),

ethylsilicate (TEOS), ascorbic acid and amino acids were purchased from Sigma-Aldrich. CoCl2·6H2O, NaClO, NaOH and other chemicals of analytical grade were attained from Sinopharm Chemical Reagent Co.,

Ltd.

(Shanghai,

China).

[3-(4,

5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-



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sulfophenyl)-2H-tetrazolium (MTS) was purchased from Promega. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin-streptomycin solution were obtained from Invitrogen. Ultrapure water, which was obtained through Merck Millipore purification system, was used to prepare solutions in all experiments. Instruments. Zetasizer Nano (Malvern) was used to measure zeta potential and hydrodynamic size of TPNPs and CoOOH. TEM results were obtained on JEM 2100 transmission electron microscope (Hitachi). One-photon fluorescence intensity was collected through a Fluoromax-4 spectrofluorometer (HORIBA JobinYvon, Edison, NJ) with a 400 µL quartz cuvette. The concentration of Hela cells was determined with the TC10TM automated cell counter (Bio-Rad). The MTS assay was operated on the Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT). Two-photon laser scanning confocal microscope (Olympus FV1000, Japan) was used to collected cell and tissue fluorescence images. Synthesis of TP-ICPES. Two-photon organic dye-modified silane monomer (TP-ICPES) was prepared following our previous work.

32

0.486 mmol of 3-ICPES and 0.435 mmol of two-photon organic dye F

(TP-F) were dissolved in 180 mL of tetrahydrofuran (THF). Then, under a dry inert atmosphere of N2, the mixed solution was heated at reflux in the dark for 24 h. At last, 30 mL of THF was added to the obtained TP-ICPES product to prepare a solution with a concentration of ～ 6 mM . Preparation of TPNPs. CTAB (0.06 g) and NaOH (3 M, 0.15 mL) were mixed in 30 mL of ultrapure water, and the mixed solution was heated to about 80 ºC. Then, 75 µL of TEOS and 0.36 mmol of TPICPES mixture were added drop by drop, and the resulting suspension kept stirring for 4 h under 80 ºC. The final solid product was collected by centrifugation and washed with ultrapure water and methanol for five times. The resulting TPNPs were dried and stored in dry environment for further use. Preparation of CoOOH nanoflakes and CoOOH-Modified TPNPs. 2 mL of a mixed aqueous solution containing 0.2 M NaClO and 0.8 M NaOH were added to 10 mM of CoCl2 (1 mL) solution, and 6 Environment ACS Paragon Plus

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the mixture was sonicated for 30 min. Then the product was collected by centrifugation, and the supernatant was kept for further use. The CoOOH-modified TPNPs was then prepared by mixing 10 mg of TPNPs with different amount (0, 1, 2, 4, 6, 8 mg) of CoOOH nanoflakes for 20 min. Detection of AA in buffer solution. CoOOH-modified TPNPs solution (100 µg/mL) was diluted by the phosphate buffered solution (50 mM PBS with 5 mM MgCl2, pH 7.4) and different concentrations (0, 1, 2, 10, 20, 50, 100, 200, 300, 500 µM) of AA were added to the solution at room temperature. After 20 min, the fluorescence spectra of the mixture were recorded on the Fluoromax-4 spectrofluorometer with a 400 µL quartz cuvette. Preparation of cell extracts. The Hela cells (1.0×106/mL cells), which were obtained from Prof. Mao Ye (Department of Biology, Hunan University), were first centrifuged for 3 min at 25 ℃ (800 rpm) with the supernatant being removed, and the cells precipitate was redispersed in 1 mL PBS buffer with 5mM MgCl2. Then, the redispersed cells were subjected to a sonication treatment (30 min with 3s on and 3s off) in an ice-water bath using a probe-type sonicator (150 W). The disrupted cell suspension was centrifuged (10000 rpm) for 15 min, and the supernatant was collected. Finally, the resulting cell extracts was stored at 4 °C for further use. Cytotoxicity of CoOOH-modified TPNPs. The Hela cells were grown in DMEM cell medium containing 10% fetal bovine serum and 1% penicillin-streptomycin and cultured in a humidified incubator at 37 oC with 5% CO2. For cell cytotoxicity assay, Hela cells were first seeded in a 96-well plate with a density of 4x 103 cells per well for overnight incubation. Then, the cell medium was replaced with 200 µL of fresh cell medium containing desired concentration of CoOOH-modified TPNPs. After 2 h, the cell medium was removed and 200 µL of fresh DMEM cell medium was added. After 48 h, 20 µL MTS and 100 µL DMEM cell medium mixture was added to each well and incubated at 37 oC with 5% CO2 for 1.5 h. The cell viability was determined by using a multimode microplate.



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Fluorescence Microscopy Imaging. To study the capability of CoOOH-modified TPNPs nanoprobe for monitoring intracellular AA levels in cancer cells, Hela cells were first seeded in a 30-mm glass bottomed dish plated and grown to around 80% confluency for 16 h before the experiment. Cells were washed three times with 1 mL PBS containing 5 mM MgCl2 and then incubated with the proper nanoprobe at the desired concentration in PBS containing 5 mM MgCl2 at 37 °C in 5% CO2. After 2 h, cells were washed three times with 1 mL PBS containing 5 mM MgCl2, dispersed in 1 mL PBS containing 5 mM MgCl2 and then subjected to confocal fluorescence microscopy analysis. Tissue imaging was also used to monitor intracellular AA levels in rat liver slice with a similar procedure. Two-photon confocal fluorescence imaging of cells and tissues were observed under an Olympus FV1000 two-photon laser scanning confocal microscope at 740 nm. RESULTS AND DISCUSSION Prepartion and Characterization of TPNPs, CoOOH nanoflakes, and CoOOH-modified TPNPs nanoprobe. In this TP nanoprobe, TPNPs acted as fluorescence reporters and were prepared by modifying the silica nanoparticles with two-photon dye. The size of TPNPs was characterized by TEM (Figure S1a), and particle size distribution of TPNPs was investigated by DLS (Figure S1b). Zeta potential on the surface of TPNPs was -13 mV. Amorphous CoOOH nanoflakes were prepared via a one-step approach and the nanostructure was analyzed through TEM (Figure 1a). The zeta potential of the CoOOH nanoflakes was 4.5 mV, showing the positively charge of the nanoflakes. Therefore, the negatively charged TPNPs could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes by electrostatic force, leading to remarkable decrease in their fluorescence intensity. The formation of CoOOH-modified TPNPs nanoprobe was also verified by DLS (Figure S1c), zeta potential measurements (0.25 mV) and TEM (Figure 1b). From the TEM results, we can see that CoOOH-modified TPNPs are slightly agglomerated. Compositional analyses with energy dispersive spectroscopy (EDS) further indicated the presence of Co and O elements in this new proposed nanoprobe (Figure S2).


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Figure 1. TEM images of CoOOH nanoflakes (a) and CoOOH-modified TPNPs(b). Optical Properties of CoOOH-modified TPNPs nanoprobe. To investigated the feasibility of the TP nanoprobe, we then studied optical properties of TPNPs, CoOOH nanoflakes and the nanoprobe, respectively. As shown in Figure S3, under excitation of 370nm, the TPNPs show strong fluorescence emission at 470 nm and the emission spectrum overlaps well with the absorbance spectrum of CoOOH, resulting in energy-transfer from the TPNPs to CoOOH nanoflakes, thereby effectively quenching onephoton and two-photon fluorescence of TPNPs. The quenching degree of the TPNPs depends on the concentration of CoOOH nanoflakes. As shown in Figure S4, a dramatic decrease of fluorescence intensity of the TPNPs was observed with an increase of the CoOOH nanoflakes concentration from 0 to 80 µg/mL, and the maximum quenching up to 96.75% was achieved. And, the most suitable weight ratio of CoOOH nanoflakes and TPNPs is 0.6, because when the weight ratio is lower than 0.6, the quenching efficiency is low, however, when the weight ratio is higher than 0.6, the fluorescence enhancement is not satisfactory (Figure S5). Moreover, for a TP bioimaging nanoprobe, the TP excitation action crosssection (Φδ) is one of the most important photophysical parameters. As shown in Figure S6, by exciting with 740 nm femtosecond pulses, the Φδ values of TPNPs was 103 GM, which was similar with free TP-F or TP-ICPES. These all optical properties of the nanoprobe indicated that our proposed hybrid nanosystem is suitable for turn-on detection and TP imaging of biological molecule in living cells and tissues



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Fluorescence Response to AA in Aqueous Solution with the Nanoprobe. The effect of pH on the fluorescent response of the nanoprobe was first investigated (Figure S7, Supporting Information). When 50 µΜ AA was added to the nanoprobe, there is no obvious change on the fluorescence enhancement from pH 6.0 to 8.0. Then, the fluorescence response to AA in buffer solutions of the nanoprobe was investigated by one-photon excited. As Figure 2a shown, a large increase of fluorescence intensity of TPNPs was observed when the AA concentration was increased from 0 to 500 µM. Figure 2b described the relationship between the fluorescence enhancement and the different concentrations of AA, and inset of Figure 2b showed a good linear correlation (R = 0.9913) from 1 to 20 µM with a LOD of 170 nM based on the 3σ/slope rule. The regression equation was F/F0 = 0.1003[AA] +1.3121. The large fluorescence enhancement of the sensing system might arise from the super quenching efficiency of CoOOH to lower the background fluorescence, which is favorable for affording a high sensitivity for AA.


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Figure 2. (a) Fluorescence response in the presence of different concentrations of AA, ranging from 0 to 500 µM. (b) Relationship between fluorescence enhancement and the target concentration. Inset shows the responses of the sensing system to AA at low concentration. F0 and F are the fluorescence intensity of the sensing system in the absence and presence of target, respectively. We next studied the kinetic performance of the oxidation-reduction reaction between AA and CoOOH-modified TPNPs nanoprobe. We added 500 µM AA to the nanoprobe solution, and then recorded fluorescence intensity change of the TNNPs as a function of time. As shown in Figure S8, the fluorescence intensity of TPNPs increased quickly with the elongation of time and reached equilibrium in 10 min, revealing a rapid decomposition of the CoOOH nanoflakes by AA. Meanwhile, during the redox reaction, AA was oxidized to generate dehydroascorbic acid as shown in eq 1. In order to prove that the reduction of CoOOH proceeds on the nanoprobe, we also study the reaction product by UV-vis spectroscopy assay (Figure S9).

Selectivity of the Nanoprobe for AA Detection. Besides sensitivity, selectivity is another important parameter to evaluate the performance of a new developed nanoprobe. Particularly, for a fluorescence nanoprobe with potential applications in practical complex samples, it is necessary that the probe possesses a highly selective response to the target over other potentially competing species, because the activity of target might be influenced by other reactive species existing in biomatrix. So, the selectivity experiments of the CoOOH-modified TPNPs nanoprobe for AA were implemented with various potentially competing interfering agents. As shown in Figure 3, little fluorescence enhancement ratio changes was observed with all other electrolytes and reducing compounds such as GSH and Gly, indicating that our proposed nanoprobe exhibited high selectivity to AA. The excellent specificity



combined with high sensitivity and fast response to AA suggests that this nanoprobe might be directly applied to detecting AA in living system. To test the feasibility of the designed two-photon nanoprobe, the recovery of spiked of AA was investigated in cell extracts samples. All the cell extracts samples showed that no AA can been detected. Different concentrations of AA (20, 100, 500 µM ) solution were spiked in these samples, and then the CoOOH-modified TPNPs were employed to detect the AA concentration. At last, its fluorescence enhancement was compared with concentrated standard AA solution. As shown from the Table 1, the results obtained in cell extracts samples show satisfactory recovery values. To further investigate the sensing ability of the nanoprobe in the real samples, the AA titration experiments in the cell extracts samples were also performed. As shown in Figure S10, the AA titration curve in the cell extracts was similar to that in the buffer solution, with a linear concentration range from 2 to 100 µM for AA, which confirmed that the proposed sensing system was applicable for practical AA detection in real samples with other potentially competing species coexisting. Table1. Recovery study of spiked AA in cell extracts with the nanoprobe. cell extracts

a

AA spiked (mM)

AA recovered meana ± SDb 20.80±0.48

recovery (%)

1

20.00

2

100.00

102.00±1.2

102

3

500.00

506.22±0.20

101

Mean of three determinations. bSD: standard deviation 14 12 10 F/F0

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8 6 4 2 0 a b c d e f g h i

j k l m n o p q r s


104

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Figure 3. Selectivity of the CoOOH-modified TPNPs nanoprobe for AA over other potential interfering agents (AA at concentration of 500 µM and all other compounds at concentration of 10 mM). a) KCl, b) NaCl, c) MgSO4, d) CaCl2, e) MnCl2, f ) Zn(NO3)2, g) Ba(NO3)2, h) CoCl2, i) Al(NO3)3, j) FeCl3, k) Cys, l) Arg, m) Trp, n) Try, o) Phe, p) Gly, q) GSH, r) NaClO, s) AA. F0 and F are the fluorescence intensities of the nanoprobe in the absence and presence of the target (AA) or nontarget samples, respectively. Cytotoxicity Investigation of the Nanoprobe. To evaluate the cytotoxicity of the nanoprobe, we performed MTS assay in Hela cells. The cells were treated with nanoprobe of different concentrations (10, 20, 40, 80, 100 and 200 µg/mL). The absorbance of MTS at 490 nm depends on the degree of activation of the cells. As shown in Figure 4, the cell viability was more than 90% when the concentration of the CoOOH-modified TPNPs nanoprobe was up to 200 µg/mL, indicating good biocompatibility of our proposed nanoprobe.

100 80 Cell Viability (%)

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60 40 20 0

10

20 40 80 100 200 Probe Concentration (µ µg/mL)

Figure 4. The viability of Hela cells treated with CoOOH-modified TPNPs nanoprobe. TP Imaging of AA in Cancer Cells. The CoOOH-modified TPNPs was then used in living cancer cells to monitor intracellular AA levels, we chose Hela cells as the model system and the results were analyzed by two-photon laser scanning confocal microscope with a 740 nm femtosecond pulses. As shown Figure 5a, Hela cells incubated with CoOOH-modified TPNPs nanoprobe (200 µg/ mL) in a PBS containing 5 mM MgCl2 buffer supplemented with 10% (v/v) cell culture medium for 2 h did not exhibit 13 Environment ACS Paragon Plus


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obvious two-photon fluorescence signal. However, Hela cells were first incubated with 200 µg/ mL of CoOOH-modified TPNPs nanoprobe in a PBS containing 5 mM MgCl2 buffer supplemented with 10% (v/v) cell culture medium for 2 h and then with 100 µM AA for another 1 h, a strong green two-photon excited fluorescence emission was observed (Figure 5b), indicating efficient uptake of nanoprobe and the ability of imaging AA in living cells. To verify that the recovery of the fluorescence was resulted from AA rather than the other substances in living cells, AA (100 µM) was treated with ascorbate oxidase (AOase) (10 U/µL) for 0.5 h to suppress the reductive properties, and the resulting product was incubated with pretreated Hela cells. As shown in Figure 5c, only very weak two-photon fluorescence could be observed. In addition, to ensure the utility of the nanoprobe for imaging AA in living cells, we took time-courses cell images using two-photon laser scanning confocal microscope. With HeLa cells treated with nanoprobe and AA, as seen from Figure 6a, the brightness of fluorescence image gradually increased with the elongation of time and reached equilibrium after 15 min. However, the Hela cells which were only treated with nanoprobe displayed a weak fluorescence signal in all posscess (Figure 6b). Taken together, these results strongly support the feasibility of intracellular AA monitoring by CoOOHmodified TPNPs.


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Figure 5. Two-photon confocal fluorescence microscopy images of Hela cells treated with (a) 200 µg/ mL CoOOH-modified TPNPs nanoprobe; (b) 200 µg/ mL CoOOH-modified TPNPs nanoprobe and 100 µM AA; (c) 200 µg/ mL CoOOH-modified TPNPs nanoprobe, AA 100 µM and 10 U/µL AOase. The green channel is two-photon fluorescence of TPNPs. Differential interference contrast (DIC) microscopy images of cells are shown in the second column. Overlap of fluorescence and differential interference contrast (DIC) images are shown in the third column. Two-photon images were collected at 450-530 nm. Scale bar = 20 µm.

Figure 6. Time-dependent two-photon confocal fluorescence microscopy images of Hela cells pretreated with 200 µg/ mL CoOOH-modified TPNPs nanoprobe, and then incubated with 100 µM AA(a); only treated with 200 µg/ mL CoOOH-modified TPNPs nanoprobe (b). TP Imaging of AA in Tissues. We next assessed the ability of the nanoprobe to image AA in tissues. The first rat liver slice was incubated with 200µg/ mL nanoprobe, while the second slice was pretreated with 200 µg/ mL nanoprobe and then treated with 100 µM AA for another 1 h. Subsequently, the twophoton fluorescence images of the nanoprobe in tissue were collected with two-photon laser scanning confocal microscope. As shown in Figure 7, the slice treated with the nanoprobe did not show obvious two-photon fluorescence (Figure 7a), while the slice which was first treated with the nanoprobe for 2h and then with 100µM AA for another 1h exhibited strong fluorescence (Figure 7b). Moreover, the changes of fluorescence signal intensity with different scan depth were also investigated by two-photon microscopy in z-scan mode. As shown in Figure S11, this new nanoprobe was able to make tissue image 15 Environment ACS Paragon Plus


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at depths of 0-180 µm. These results fully demonstrated that CoOOH-modified TPNPs nanoprobe has excellent staining ability and tissue penetration, which making them suitable for effective imaging of cellular AA at tissue level.

Figure 7. Two-photon imaging of a rat liver frozen slice treated withCoOOH-modified TPNPs (a) and CoOOH-modified TPNPs and AA (b), respectively. Two-photon images were collected at 450-530 nm. Scale bars are 100 µm CONCLUSIONS In summary, we have demonstrated a novel two-photon nanoprobe for fluorescence “turn-on” detection and imaging of AA in living cells and tissues. In this nanosystem, the negatively charged twophoton nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with twophoton dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge by electrostatic force, leading to remarkable decrease in their fluorescence intensity. However, the CoOOHinduced quenching effect could be reversed in the presence of AA, since it efficiently reduces CoOOH into Co2+, and results in the destruction of the CoOOH nanoflakes, which affords a highly sensitive detection of AA in aqueous solutions with a LOD of 170 nM. The nanoprobe also shows high selectivity toward AA over common potential interfering species. By taking advantages of two-photon imaging technology such as high spatial localization and deep imaging depth, and excellent membranepermeability and good biocompatibility of the silica nanoparticles, the nanoprobe was then applied for 16 Environment ACS Paragon Plus

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two-photon fluorescence imaging of AA in living cells and liver tissues with satisfactory results. We anticipate this quenching and recovery strategy might provide a new platform for efficient monitoring targets of interest in vivo.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author *Email: [email protected]; [email protected] Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGEMENT This work was supported by the National Key Scientific Program of China (2011CB911000), the National Key Basic Research Program of China (2013CB932702), NSFC (Grants 21505032, 21325520, 21327009, 21177036), the Foundation for Innovative Research Groups of NSFC (Grant 21221003), the Key scientific research project of higher education of the Henan province (16A150013,15A150013, 15A150016), the National Instrumentation Program (2011YQ030124). REFERENCES (1) Janda, P.; Weber, J.; Dunsch, L.; Lever, A. B. P.; Chem., A. Anal. Chem. 1996, 68, 960-965. 17 Environment ACS Paragon Plus


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For TOC only:


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Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection

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