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

Article abstract of DOI:10.1021/acs.analchem.6b01352

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, two-photon (TP) excited probes are more suitable for bioimaging, 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 a two-photon dye, could adsorb cobalt oxyhydroxide (CoOOH) nanoflakes which carried positive charge by electrostatic force, leading to a remarkable decrease in their fluorescence intensity. However, the introduction of AA could induce the fluorescence recovery of the nanoprobe because it could reduce CoOOH into Co²⁺ and result in the destruction of the CoOOH nanoflakes. The nanosystem exhibits a high sensitivity toward 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.

Full text of DOI:10.1021/acs.analchem.6b01352

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Article
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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Page 1 of 20
Analytical Chemistry
Efficient Two-Photon Fluorescence Nanoprobe for Turn-On Detection
and Imaging of Ascorbic Acid in Living Cells and Tissues
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†
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HongꢀMin Meng , XiaoꢀBing Zhang* , Chan Yang , Hailan Kuai , GuoꢀJiang Mao , Liang Gong ,
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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.
xbzhang@hnu.edu.cn；cjb@htu.edu.cn
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ABSTRACT:
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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, two-
photon (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
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(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
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fluorescence recovery of the nanoprobe. Since it could reduce CoOOH into Co , 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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Analytical Chemistry
INTRODUCTION
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Monitoring the distribution of reactive species in living systems is of great importance for
understanding their physiological functions and pathological effects, as well as earlyꢀstage 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
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against oxidative damages and involvement in neurotransmitterꢀrelated enzymes. In addition, a variety
of epidemiologic studies and clinical trials have shown that the abnormal level of AA is associated with
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many diseases, such as scurvy, depression, connective tissue defects and diarrhea. Therefore, the
development of efficient methods for monitoring AA level in living systems has become an important
subject of current chemical research.
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Up to now, several methods including chromatography, electrochemical analysis,
and
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fluorescence assay
have been developed for the detection of AA in vitro or in vivo. For the
bioimaging of AA in living systems, fluorescent probeꢀbased imaging technique might be an ideal
candidate due to its intrinsic advantages such as high sensitivity, realꢀtime and inꢀsitu monitoring and
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nonꢀsample damaging.
So far, several organic moleculeꢀbased fluorescent probes have been reported
However, there are several obstacles, such as, photobleaching, poor membraneꢀ
permeability and waterꢀsolubility, which strictly limit their biological applications.
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for AA assay.
Various nanomaterials, such as quantum dots, MnO nanosheets, and carbon nanomaterials, which
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possess good membraneꢀpermeability and excellent biocompatibility, have also been employed for
designing of fluorescent probes for sensing AA in the past decade. Yan group developed a CdTe QDꢀ
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based fluorescent nanoprobe for selectively detecting AA in biological fluids. By absorbing 7ꢀ
hydroxycoumarin on singleꢀlayer MnO nanosheets, Mao group reported a new fluorescent nanoprobe
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for in vivo sensing of AA in rat brain. Based on the specific oxidationꢀreduction reaction between
CoOOH nanoflakes and AA, Lin et al. reported CoOOH nanoflakesꢀmodified carbon dotsꢀbased
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fluorescent nanoprobe for monitoring cerebral AA in brain microdialysate. Although they are useful,
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these probes are based on traditional oneꢀphoton excited technology with short excitation wavelengths,
which suffered from photobleaching, interference from bioꢀrelated 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 nanoflakeꢀmodified persistent luminescence nanoparticle as nanoprobe
for detection and imaging of AA in living cells and in vivo, which permits detection and imaging of AA
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without external excitation, and is free from bioꢀrelated autofluorescence interference.
Twoꢀphoton fluorescent microscopy (TPM), which employed nearꢀinfrared laser pulses for
excitation, afforded a number of advantages over traditional oneꢀphoton 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
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imaging resolution, better threeꢀdimensional spatial localization and deeper penetration depth.
These advantages make TP fluorescence probe more suitable for bioꢀimaging applications. Recently,
many TP probes based on organic fluorophores or nanomaterials have been developed for detection and
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bioimaging of metal ions, anions and neutral molecules.
Among them, nanoprobes are much more
feasible for intracellular imaging owing to their nanoꢀendocytosis effect induced good membraneꢀ
permeability. To our best knowledge, no twoꢀphoton probe has been reported for AA detection up to
date. In this work, by combining twoꢀphoton nanoparticles (TPNPs) with CoOOH nanoflakes, we try to
develop a twoꢀphoton fluorescence nanoprobe (CoOOHꢀModified 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 twoꢀphoton
nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with twoꢀphoton 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
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Analytical Chemistry
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
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Co , and the fluorescence of TPNPs is remarkably restored due to the decomposion of the CoOOH
nanoflakes. The twoꢀphoton 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 membraneꢀpermeability and good biocompatibility. At
last, the nanosystem is applied for TP excited fluorescence imaging of AA in living cells and tissues
with satisfactory result.
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Scheme 1. Preparation of CoOOHꢀModified TPNPs (A) and Detection and Twoꢀphoton imaging of
Ascorbic Acid in Living Cells and Tissues through CoOOHꢀModified TPNPs (B).
EXPERIMENTAL SECTION
Reagents.
Hexadecyltrimethylammonium bromide (CTAB), 3ꢀisocyanatopropyltriethoxysilane (3ꢀICPES),
ethylsilicate (TEOS), ascorbic acid and amino acids were purchased from SigmaꢀAldrich. CoCl ·6H O,
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specified,
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NaClO, NaOH and other chemicals of analytical grade were attained from Sinopharm Chemical Reagent
Co., Ltd. (Shanghai, China). [3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀcarboxymethoxyphenyl)ꢀ2ꢀ(4ꢀ
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sulfophenyl)ꢀ2Hꢀtetrazolium (MTS) was purchased from Promega. Dulbecco’s modified Eagle’s
medium (DMEM), fetal bovine serum (FBS) and penicillinꢀstreptomycin solution were obtained from
Invitrogen. Ultrapure water, which was obtained through Merck Millipore purification system, was used
to prepare solutions in all experiments.
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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). Oneꢀphoton fluorescence intensity was collected through a Fluoromaxꢀ4 spectrofluorometer
(HORIBA JobinYvon, Edison, NJ) with a 400 ꢀL quartz cuvette. The concentration of Hela cells was
determined with the TC10TM automated cell counter (BioꢀRad). The MTS assay was operated on the
Synergy 2 MultiꢀMode Microplate Reader (BioꢀTek, Winooski, VT). Twoꢀphoton laser scanning
confocal microscope (Olympus FV1000, Japan) was used to collected cell and tissue fluorescence
images.
Synthesis of TP-ICPES. Twoꢀphoton organic dyeꢀmodified silane monomer (TPꢀICPES) was prepared
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following our previous work. 0.486 mmol of 3ꢀICPES and 0.435 mmol of twoꢀphoton organic dye F
TPꢀF) 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 TPꢀICPES 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 ꢀL of TEOS and 0.36 mmol of TPꢀ
ICPES 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 CoCl (1 mL) solution, and
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Analytical Chemistry
the mixture was sonicated for 30 min. Then the product was collected by centrifugation, and the
supernatant was kept for further use. The CoOOHꢀmodified 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.
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Detection of AA in buffer solution. CoOOHꢀmodified TPNPs solution (100 ꢁg/mL) was diluted by the
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phosphate buffered solution (50 mM PBS with 5 mM MgCl , pH 7.4) and different concentrations (0, 1,
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, 10, 20, 50, 100, 200, 300, 500 ꢁM) of AA were added to the solution at room temperature. After 20
min, the fluorescence spectra of the mixture were recorded on the Fluoromaxꢀ4 spectrofluorometer with
a 400 ꢀL quartz cuvette.
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Preparation of cell extracts. The Hela cells (1.0×10 /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
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mM MgCl . Then, the redispersed cells were subjected to a sonication treatment (30 min with 3s on
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and 3s off) in an iceꢀwater bath using a probeꢀtype 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% penicillinꢀstreptomycin and cultured in a humidified
o
incubator at 37 C with 5% CO . For cell cytotoxicity assay, Hela cells were first seeded in a 96ꢀwell
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plate with a density of 4x 10 cells per well for overnight incubation. Then, the cell medium was
replaced with 200 ꢁL of fresh cell medium containing desired concentration of CoOOHꢀmodified
TPNPs. After 2 h, the cell medium was removed and 200 ꢁL of fresh DMEM cell medium was added.
After 48 h, 20 ꢀL MTS and 100 ꢀL DMEM cell medium mixture was added to each well and incubated
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at 37 C with 5% CO 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 CoOOHꢀmodified TPNPs nanoprobe for
monitoring intracellular AA levels in cancer cells, Hela cells were first seeded in a 30ꢀmm glass
bottomed dish plated and grown to around 80% confluency for 16 h before the experiment. Cells were
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washed three times with 1 mL PBS containing 5 mM MgCl and then incubated with the proper
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cells were washed three times with 1 mL PBS containing 5 mM MgCl , dispersed in 1 mL PBS
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containing 5 mM MgCl and then subjected to confocal fluorescence microscopy analysis. Tissue
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imaging was also used to monitor intracellular AA levels in rat liver slice with a similar procedure.
Twoꢀphoton confocal fluorescence imaging of cells and tissues were observed under an Olympus
FV1000 twoꢀphoton 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 twoꢀphoton 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
oneꢀstep 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 CoOOHꢀmodified
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 CoOOHꢀmodified 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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Analytical Chemistry
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Figure 1. TEM images of CoOOH nanoflakes (a) and CoOOHꢀmodified 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 energyꢀtransfer from the TPNPs to CoOOH nanoflakes, thereby effectively quenching oneꢀ
photon and twoꢀphoton 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
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0 ꢀg/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 crossꢀ
section (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
TPꢀF or TPꢀICPES. These all optical properties of the nanoprobe indicated that our proposed hybrid
nanosystem is suitable for turnꢀon 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
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fluorescent response of the nanoprobe was first investigated (Figure S7, Supporting Information). When
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from pH 6.0 to 8.0. Then, the fluorescence response to AA in buffer solutions of the nanoprobe was
investigated by oneꢀphoton 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 ꢁM. 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 ꢁM with a LOD of 170 nM
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CoOOH to lower the background fluorescence, which is favorable for affording a high sensitivity for
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Figure 2. (a) Fluorescence response in the presence of different concentrations of AA, ranging from 0 to
00 ꢀM. (b) Relationship between fluorescence enhancement and the target concentration. Inset shows
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the responses of the sensing system to AA at low concentration. F and F are the fluorescence intensity
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We next studied the kinetic performance of the oxidationꢀreduction reaction between AA and
CoOOHꢀmodified TPNPs nanoprobe. We added 500 ꢀM 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 UVꢀvis
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 CoOOHꢀmodified 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
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combined with high sensitivity and fast response to AA suggests that this nanoprobe might be directly
applied to detecting AA in living system.
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To test the feasibility of the designed twoꢀphoton 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 ꢁM ) solution were spiked in these samples, and then the
CoOOHꢀmodified 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 ꢁM for AA, which
confirmed that the proposed sensing system was applicable for practical AA detection in real samples
with other potentially competing species coexisting.
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Table1. Recovery study of spiked AA in cell extracts with the nanoprobe.
a
b
cell extracts
AA spiked (mM)
AA recovered mean ± SD
recovery (%)
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Figure 3. Selectivity of the CoOOHꢀmodified TPNPs nanoprobe for AA over other potential interfering
agents (AA at concentration of 500 ꢀM and all other compounds at concentration of 10 mM). a) KCl, b)
NaCl, c) MgSO , d) CaCl , e) MnCl , f ) Zn(NO ) , g) Ba(NO ) , h) CoCl , i) Al(NO ) , j) FeCl , k) Cys,
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l) Arg, m) Trp, n) Try, o) Phe, p) Gly, q) GSH, r) NaClO, s) AA. F and F are the fluorescence intensities
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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 ꢀg/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 CoOOHꢀmodified TPNPs nanoprobe was up to 200 ꢀg/mL, indicating good
biocompatibility of our proposed nanoprobe.
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Probe Concentration (ꢀg/mL)
Figure 4. The viability of Hela cells treated with CoOOHꢀmodified TPNPs nanoprobe.
TP Imaging of AA in Cancer Cells. The CoOOHꢀmodified 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 twoꢀphoton laser scanning confocal microscope with a 740 nm femtosecond pulses. As
shown Figure 5a, Hela cells incubated with CoOOHꢀmodified TPNPs nanoprobe (200 ꢀg/ mL) in a PBS
containing 5 mM MgCl buffer supplemented with 10% (v/v) cell culture medium for 2 h did not exhibit
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obvious twoꢀphoton fluorescence signal. However, Hela cells were first incubated with 200 ꢀg/ mL of
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CoOOHꢀmodified TPNPs nanoprobe in a PBS containing 5 mM MgCl buffer supplemented with 10%
2
(v/v) cell culture medium for 2 h and then with 100 ꢁM AA for another 1 h, a strong green twoꢀphoton
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 ꢁM) was treated with ascorbate
oxidase (AOase) (10 U/ꢁL) 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 twoꢀphoton fluorescence
could be observed. In addition, to ensure the utility of the nanoprobe for imaging AA in living cells, we
took timeꢀcourses cell images using twoꢀphoton 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 CoOOHꢀ
modified TPNPs.
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Figure 5. Twoꢀphoton confocal fluorescence microscopy images of Hela cells treated with (a) 200 ꢀg/
mL CoOOHꢀmodified TPNPs nanoprobe; (b) 200 ꢀg/ mL CoOOHꢀmodified TPNPs nanoprobe and 100
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M AA; (c) 200 ꢀg/ mL CoOOHꢀmodified TPNPs nanoprobe, AA 100 ꢁM and 10 U/ꢁL AOase. The
green channel is twoꢀphoton 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. Twoꢀphoton images were collected at 450ꢀ530 nm.
Scale bar = 20 ꢁm.
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Figure 6. Timeꢀdependent twoꢀphoton confocal fluorescence microscopy images of Hela cells
pretreated with 200 ꢀg/ mL CoOOHꢀmodified TPNPs nanoprobe, and then incubated with 100 ꢁM
AA(a); only treated with 200 ꢀg/ mL CoOOHꢀmodified 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 200ꢀg/ mL nanoprobe, while the second slice was pretreated
with 200 ꢀg/ mL nanoprobe and then treated with 100 ꢁM AA for another 1 h. Subsequently, the twoꢀ
photon fluorescence images of the nanoprobe in tissue were collected with twoꢀphoton laser scanning
confocal microscope. As shown in Figure 7, the slice treated with the nanoprobe did not show obvious
twoꢀphoton fluorescence (Figure 7a), while the slice which was first treated with the nanoprobe for 2h
and then with 100ꢀM AA for another 1h exhibited strong fluorescence (Figure 7b). Moreover, the
changes of fluorescence signal intensity with different scan depth were also investigated by twoꢀphoton
microscopy in zꢀscan mode. As shown in Figure S11, this new nanoprobe was able to make tissue image
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at depths of 0ꢀ180 ꢀm. These results fully demonstrated that CoOOHꢀmodified TPNPs nanoprobe has
excellent staining ability and tissue penetration, which making them suitable for effective imaging of
cellular AA at tissue level.
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Figure 7. Twoꢀphoton imaging of a rat liver frozen slice treated withCoOOHꢀmodified TPNPs (a) and
CoOOHꢀmodified TPNPs and AA (b), respectively. Twoꢀphoton images were collected at 450ꢀ530 nm.
Scale bars are 100 ꢀm
CONCLUSIONS
In summary, we have demonstrated a novel twoꢀphoton nanoprobe for fluorescence “turnꢀon”
detection and imaging of AA in living cells and tissues. In this nanosystem, the negatively charged twoꢀ
photon nanoparticles (TPNPs), which were prepared by modifying the silica nanoparticles with twoꢀ
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 CoOOHꢀ
induced quenching effect could be reversed in the presence of AA, since it efficiently reduces CoOOH
2
+
into Co , 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 twoꢀphoton imaging
technology such as high spatial localization and deep imaging depth, and excellent membraneꢀ
permeability and good biocompatibility of the silica nanoparticles, the nanoprobe was then applied for
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Analytical Chemistry
twoꢀphoton 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.
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
AUTHOR INFORMATION
Corresponding Author
*
Email: xbzhang@hun.edu.cn; cjb@htu.edu.cn
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,
2
1327009, 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).
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