Near Infrared Graphene Quantum Dots-Based Two-Photon

Mar 10, 2017 - Therefore, the development of effective methods for monitoring the AA level in living systems is of great significance. ... impressive ...
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Near Infrared Graphene Quantum Dots-Based Two-Photon Nanoprobe for Direct Bioimaging of Endogenous Ascorbic Acid in Living Cells Li-Li Feng,† Yong-Xiang Wu,*,† Dai-Liang Zhang,† Xiao-Xiao Hu,† Jing Zhang,† Peng Wang,† Zhi-Ling Song,*,‡ Xiao-Bing Zhang,*,† and Weihong Tan† †

Molecular Science and Biomedicine Laboratory, State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Collaborative Innovation Center for Chemistry and Molecular Medicine, Hunan University, Changsha 410082, China ‡ Key Laboratory of Sensor Analysis of Tumor Marker, Ministry of Education, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao 266042, China S Supporting Information *

ABSTRACT: Ascorbic acid (AA), as one of the most important vitamins, participates in various physiological reactions in the human body and is implicated with many diseases. Therefore, the development of effective methods for monitoring the AA level in living systems is of great significance. Up to date, various technologies have been developed for the detection of AA. However, few methods can realize the direct detection of endogenous AA in living cells. In this work, we for the first time reported that near-infrared (NIR) graphene quantum dots (GQD) possessed good two-photon fluorescence properties with a NIR emission at 660 nm upon exciting with 810 nm femtosecond pulses and a two-photon (TP) excitation action cross-section (δΦ) of 25.12 GM. They were then employed to construct a TP nanoprobe for detection and bioimaging of endogenous AA in living cells. In this nanosystem, NIR GQDs (NGs), which exhibited lower fluorescence background in living system to afford improved fluorescence imaging resolution, were acted as fluorescence reporters. Also CoOOH nanoflakes were chosen as fluorescence quenchers by forming on the surface of NGs. Once AA was introduced, CoOOH was reduced to Co2+, which resulted in a “turnon” fluorescence signal of NGs. The proposed nanoprobe demonstrated high sensitivity toward AA, with the observed LOD of 270 nM. It also showed high selectivity to AA with excellent photostability. Moreover, the nanoprobe was successfully used for TP imaging of endogenous AA in living cells as well as deep tissue imaging.

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chromatography,11 capillary electrophoresis,12 electrochemistry,13 colorimetry,14 and fluorescence spectroscopy.15,16 Among them, the fluorescent assay technique received extensive attention because of the advantages of high sensitivity, good reproducibility, easy to realize real-time, and in situ monitoring.17 Recently, several organic molecule-based fluorescent probes have been developed for the AA assay.18−20 Although these probes have achieved impressive results, some drawbacks still exist, for instance, easy photobleaching, poor photostability, and difficult separation, which limited their further application in living systems. Recently, a variety of nanomaterials, including quantum dots, Au nanoclusters, and CoOOH flakes have been utilized for the

scorbic acid (AA), or vitamin C, as one of the most important vitamins to maintain the body’s normal physiological function, participates in various reactions in the body. More specifically, AA as a very important component of the endogenous environment plays an important role of enzyme cofactor and a component of the enzyme which related to the neurotransmitters.1−4 AA is also capable of antioxygenation and resists the cells damage from free radical, effectively avoiding cytometaplasia.5,6 Meanwhile, AA can promote the synthesis of collagen in the body, increasing the body resistance.7 Research showed that the level of AA was associated with many diseases in the body, such as arteriosclerosis, scurvy, anemia, cardiovascular disease, and so on.8−10 In this regard, the development of effective methods for monitoring AA level in living systems is of great significance. Up to date, various technologies and methods have been developed for the detection of AA in vitro or in vivo, such as © 2017 American Chemical Society

Received: December 13, 2016 Accepted: March 10, 2017 Published: March 10, 2017 4077

DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084

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Analytical Chemistry Scheme 1. Schematic Illustration of the Design and Principle for AA Detection Using CoOOH-NGs

GQDs (NGs) with CoOOH nanoflakes, we for the first time developed a fluorescence nanoprobe with TP excitation and NIR emission features for directly detection of endogenous AA in living cells and deep tissue imaging. In this nanosystem, the NGs with TP excitation fluorescence feature acted as fluorescence reporters and the CoOOH nanoflakes formed on the surface of NGs were served as fluorescence quenchers (Scheme 1). The CoOOH nanoflakes can efficiently quench the fluorescence of the NGs by energy transfer (ET), for the absorption spectrum of the CoOOH nanoflakes overlaps well with the emission spectrum of NGs. In the presence of AA, the CoOOH was reduced to Co2+ by AA, resulting in the decomposition of the CoOOH nanoflakes and the termination of ET process and leading to fluorescence recovery of the NGs. In this nanosystem, the NGs showed excellent TP absorption property and possessed a δΦ value up to 25.12 GM. The nanoprobe exhibited high sensitivity and selectivity to AA as well as excellent biocompatibility, making it a promising probe for imaging of endogenous AA in living cells and for deep tissue imaging.

sensing of AA. An’s group presented an “off−on” approach for the detection of AA using carbon dots as a fluorescent probe.21 On the basis of protein-modified Au nanoclusters, Lv’s group proposed a fluorescent “turn-off” sensor to detect AA.22 On the basis of the specific reaction between CoOOH and AA, Tang’s group using CoOOH modified persistent luminescence nanoparticles (Sr2MgSi2O7, 1%; Eu, 2% Dy) for determination and screening of AA in living cells and in vivo.23 Although these probes are effective for detection of AA in vitro, most of them showed fluorescence emissions in the UV or visible light region, suffering from the limited penetration depth in tissue imaging. Therefore, it is urgent to design effective and convenient fluorescent nanoprobes which own low fluorescent background and deep tissue penetration depth for the detection of AA. Two-photon (TP) fluorescent probes with advantages such as low background signal, reduced photobleaching, deep tissue penetration depth, and low phototoxicity have received much attention for their applications in the field of biomedical imaging and biosensing.24,25 By taking the advantage of TP fluorescent probes, our group recently designed an efficient TP fluorescent probe for “turn-on” detection and imaging of AA in living cells and tissues.26 However, this TP fluorescent probe cannot achieve the detection of endogenous AA in living cells, so we hope to develop a TP fluorescence probe with higher sensitivity to detect endogenous AA and to achieve deeper tissue penetration depth. It is worth noting that that both light absorption and light scattering caused by cellular component and tissues are detrimental to excitation and emission light. Particularly, the decay of the outgoing light lead to the reduction of fluorescence signal and reduced discrimination of signal from the background. Recently, NIR materials have attracted considerable attention on account of their excellent properties, such as deep issue penetration, weak scattering of emission light, and lower fluorescence background, which can reduce autofluorescence with improved signal-to-noise ratio for targets.27−30 Up to now, a number of NIR materials have been synthesized and used for biological samples detection in tissue or in vivo.31,32 Among these NIR materials, semiconductor quantum dots (QDs) owning novel electronic, magnetic, and optical properties are widely used.33,34 However, the latent toxicity of the heavy metals contained in the semiconductor QDs limits its applications in the biosystem.35−37 Actually, carbon quantum dots (CQDs), especially graphene quantum dots (GQDs), are ideal NIR materials for their admirable features such as nonblinking fluorescence emission, high cell permeability, well water solubility, and good biocompatibility.38−41 However, GQDs possessing good TP fluorescence properties with excitation and emission wavelength both in the NIR region has rarely been reported so far. In this work, combining NIR

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EXPERIMENTAL SECTION

Materials and Instruments. N,N-Dimethyldodecylamine, 4-bromobenzyl bromide, citric acid (CA), branched poly(ethylenimine) (BPEI), CH2Cl2, CH3OH, Pd(PPh3)4, Na2CO3, FeCl3, MgSO4, CHCl3, NaOH, NaClO, CoCl2·6H2O, ascorbic acid, amino acids, [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS), 2(N-morpholino) ethanesulfonic Acid (MES), Dulbecco’s modified Eagle’s medium (DMEM), 4-(2-hydroxyethyl)-1piperazineethanesulfonic acid (HEPES), tris(hydroxymethyl)aminomethane (Tris), N-ethylmaleimide (NEM), radio immunoprecipitation assay (RIPA) lysis buffer and other chemicals were analytical grade and used without further purification. Ultrapure water used in all experiments was obtained through Milli-Q system (Millipore). The transmission electron microscope (TEM) results were obtained on JEM 2100 transmission electron microscope (Hitachi). The X-ray photoelectron spectroscopy (XPS) dates was obtained on an Xray photoelectron spectroscope (Thermo Fisher Scientific). The energy dispersive spectroscopy (EDS) were collected on a field emission scanning electron microscope (SEM, JSM-6700F, Japan). Zeta-sizer Nano (Malvern) was used to measure hydrodynamic size and zeta potential CoOOH-NGs. The UV− vis absorption spectra were collected on a UV-2450 UV−vis spectrometer (Shimadzu, Japan). All the pH was performed with a Mettler-Toledo Delta 320 pH meter. Absorbance was measured on the Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT) in the MTS assay. One-photon fluorescence intensity was carried out on a Fluoromax-4 4078

DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084

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Analytical Chemistry

Figure 1. TEM image of NGs (a) and CoOOH-NGs (b).

at the Fluoromax-4 spectrofluorometer with an excitation of 480 nm. Detection of AA in Cell Extracts. Hela cells were seeded into the 6 cm cell culture dish and incubated for 48 h. Next, the DMEM medium was removed and the cell was washed with 1 mL PBS for two times. Then, 200 μL RIPA lysis buffer was added to the cell culture dish and responded for 10 min in the shaking table. After that, the disrupted cell was wiped from the cell culture dish and transferred the crushed cell suspension to 1.5 mL centrifuge tube. The crushed cell suspension was centrifuged (14 000 rpm) for 15 min, and the supernatant was collected for further use. MTS Assay. The Hela cells were grown in complete DMEM medium (with 10% fetal bovine serum and 1% penicillin− streptomycin) at 37 °C in a humidified incubator containing 5% CO2. The MTS assay was used to assess the cytotoxic effects of the CoOOH-NGs. Hela cells were seeded into a 96well microtiter plate with a density of 104 cell/well and then incubated for 24 h. Next, the original cell medium was removed and added 200 μL of fresh cell medium containing different concentrations of CoOOH-NGs. After incubating the cells for 48 h, 100 μL of cell medium and 20 μL of MTS were added to each well and incubated for 0.5 h. A multimode microplate was used to determine the cell viability. Imaging of AA in Living Cells and Tissue. Hela cells were seeded in a 30 mm optical culture dish and cultured for 24 h. After that the cells were washed three times with 1 mL of PBS buffer and then incubated with the DMEM culture medium containing 200 μg/mL nanoprobe at 37 °C in 5% CO2. After 2.5 h of incubation, the cells were washed three times with PBS buffer and then added another 1 mL of PBS buffer for subsequently TP imaging. For the tissue imaging, two pieces of rat liver slice were incubated with 200 μg/mL CoOOH-NGs for 2.5 h, then 200 μM AA was added to one of the rat liver slice and incubated for another 1 h. Next, the onephoton and TP images of AA was observed on an Olympus FV1000-MPE, and TP images were performed with a modelocked titanium-sapphire laser source set at a wavelength of 810 nm. Similar procedure was also used to the tissue imaging of CQDs. The TP imaging tissue was also performed by the Olympus FV1000-MPE with excitation at 810 nm.

spectro-fluorometer (Horiba JobinYvon, Edison, NJ). TP fluorescence images of cell and tissue were carried on a multiphoton laser scanning confocal microscope (Olympus FV1000-MPE, Japan). Synthesis of CQDs and NGs. The CQDs was synthesized as reported by previous report by Chi’s group.42 Briefly, the BPEI-modified CQDs were synthesized by pyrolyzing the mixture of CA and BPEI. In the experiment, 1.0 g of CA and 0.5 g of BPEI were dissolved with 10 mL of water in a 25 mL beaker and then heated moderately using a heating mantle (with the temperature of 200 °C). Within 20 min, most water was evaporated, leaving uniform pale-yellow gel. Next, before the gel was scorched, 1 mL of water was added with continuous heating the gel. This step was repeated about 10 times until the color of the gel turned to orange, indicating the formation of CQDs. Eventually, the obtained CQDs were adjusted to 5 mL of solution using ultrapure water and purified by silica gel column chromatography with 0.01 M HCl solution as the developing solvent. The NGs nanoparticles were first prepared by hydrothermal treatment of polythiophene (PT2), which was synthesized by previous reports.43,44 In a typical synthesis of NGs, 15 mg of PT2 was dispersed in 20 mL of NaOH solution (0.4 mM). Following, the obtained mixture was sonicated for 0.5 h and then transferred into a high pressure reactor and reacted at 170 °C for 24 h. When the reaction was accomplished and the autoclave was cooled to the room temperature, the NGs was collected. By using 0.22 mm filtering membranes, the large particles were removed. The residual NaOH was removed by dialysis against deionized water for several times. The finally obtained NGs were dispersed in water for further use. Preparation of CoOOH Nanoflakes and CoOOH-NGs. A 100 μL water solution of the NGs (1 mg/mL) was added to a 1.5 mL microcentrifuge tube containing different amounts of CoCl2 (10 mM, 0−350 μL). Then a 100 μL sample of NaClO (0.2 M) and100 μL of NaOH (0.8 M) were added to the above solution. The obtained mixture was sonicated for 10 min. Following, the CoOOH modified NGs were collected via centrifugation, washed three times with deionized water, and redispersed in water for further use. In the next experiment, 300 μL of CoCl2 was chosen as an example volume to form the CoOOH-NGs. Detection of AA in Buffer Solution. The 20 μL volume of freshly prepared CoOOH-NGs (1.0 mg/mL) was added to 200 μL of microcentrifuge tubes and then added phosphate buffered solution (20 mM PBS, pH 7.4). After that, different concentrations of AA were added to the resulting solution and obtained a final volume of 100 μL. After 20 min, the fluorescence spectra of the resulting solutions were measured

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RESULTS AND DISCUSSION Preparation and Characterization of NGs and CoOOHNGs Nanoprobe. To investigate the size and morphology of the NGs, a TEM assay was performed. As shown in Figure 1a, the diameters of NGs ranged from 2 to 7 nm with excellent dispersibility. As shown in the Figure S1A, the data of HTEM of NGs shows these NGs have a lattice. The formation of 4079

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Analytical Chemistry

As shown in Figure S12, in different media (H2O, PBS, MES, HEPES, and Tris-HCl) the fluorescence intensity of the nanoprobe (1 mg/mL, with 200 μM AA added) did not show obvious changes, indicating the stability of the nanoprobe in different conditions. Next, we investigated the sensing of the CoOOH-NGs system for AA activity. As increased concentration of AA (from 0 μM to 800 μM) was added to the solutions, the fluorescence intensity of the nanoprobe was gradually increased (Figure 2a). At low concentrations (0−800

CoOOH-NGs nanoprobe was confirmed by TEM, and the diameters of CoOOH-NGs were less than 200 nm with well dispersibility, as shown in Figure 1b. The TEM image of AA reacted CoOOH-NGs was also measured and shown in Figure S1B. The TEM image of AA reacted CoOOH-NGs indicated that the CoOOH nanoflake was completely reacted with AA. The particle size distribution of CoOOH-NGs was investigated by DLS, and from Figure S2a we can see the particle size distribution more uniform. The apparent zeta potential of the NGs and CoOOH-NGs were, respectively, 5.15 mV and 19.6 mV, showing the positive charge of the NGs and the CoOOHNGs nanoprobe (Figure S2b,c). When 800 μM AA was added, the apparent zeta potential of the solution is close to neutral, as shown in Figure S2d. As shown in Figure S3, EDS of the NGs, CoOOH-NGs, and AA reacted CoOOH-NGs were measured, and the peak of Co element demonstrated the formation of CoOOH-NGs and the reaction between CoOOH-NGs and AA. As seen from the high resolution Co (2p1/2) and Co (2p3/2) XPS spectrum of the NGs and the CoOOH-NGs, the CoOOH-NGs have obvious Co (2p1/2) and Co (2p3/2) XPS spectra, indicating that the CoOOH and NGs are linked (Figure S4). We further investigated the stability and dispersibility of our particles. In the experiment, we placed the particles for 2 days and compared with the refresh-prepared particles. As shown in the Figure S5, the particle has no obvious aggregation after being placed for 2 days. Optical Properties of CoOOH-NGs Nanoprobe. To investigated the feasibility of the composite nanoprobe, we studied the optical properties of the independent materials and the nanoprobe. As we can see from Figure S6, under excitation of 480 nm, the NGs show strong fluorescence emission at 660 nm and the emission spectrum overlaps with the UV−vis absorption spectrum of CoOOH nanoflakes. The result reveals that the ET from the NGs to CoOOH nanoflakes is feasible, suggesting the effectively quenching one-photon and TP fluorescence of NGs. To verify the reduction of CoOOH occurs on the nanoprobe, the reaction product was confirmed by UV−vis spectroscopy assay (Figure S7). As seen from Figure S7, when AA was added to the nanoprobe, the absorption peak of CoOOH disappeared. In this redox reaction, AA was oxidized to generate dehydroascorbic acid and the reaction product had a strong absorption peak at 200−300 nm. The result suggested that the reduction of CoOOH proceeds on the nanoprobe. As shown in Figure S8, the NGs fluorescence intensity decreased with an increased concentration of the CoCl2 in the nanoprobe, and the maximum quenching efficiency can reach as high as 97%. The fluorescence spectrumof NGs and CoOOH-NGs excited by 810 nm laser was further measured (Figure S9). Moreover, we measured the TP excitation action cross-section (δΦ) of the NGs in the nanoprobe. As shown in Figure S10, by exciting with 810 nm femtosecond pulses, the δΦ values of the NGs was 25.12 GM and the maximum δΦ value of the CoOOHNGs is estimated to be 2.88 GM at 810 nm, indicating that the nanoprobe is appropriate for detection AA in living cells and tissues. Fluorescence Stability of the Nanoprobe and the Response for AA. The stability of the nanoprobe at different pH was first investigated (Figure S11). The fluorescence intensity of the nanoprobe was almost constant from pH 6.8 to 8.4 when 200 μM AA was added, suggesting that the nanoprobe was stable in different pH conditions. We also investigated the stability of the nanoprobe in different media.

Figure 2. (a) Fluorescence response of CoOOH-NGs (1 mg/mL) in the presence of different concentrations of AA, ranging from 0 to 800 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.

μM) of AA, intensity of the NGs emission at 660 nm exhibited a relationship to the AA concentration (Figure 2b). The inset of Figure 2b showed a line correction between the fluorescence intensity of the nanoprobe and the AA concentrations, ranging from 1 to 30 μM. The calibration equation was (F − F0)/F0 = 0.0724[AA] + 0.7041, with a correlation coefficient of R2 = 0.9912. According to the 3σ rule, the detection limit of AA was calculated to be 270 nM. As shown in Figure S13, the kinetic studies suggested that once the AA (with the concentration of 800 μM) was added to the solution the fluorescence intensity of the nanoprobe was instantly increased and reached a stable value in a few minutes. The above results indicate that the recovery of fluorescence of NGs was caused by the reduction of CoOOH to Co2+ by AA, resulting in the release of the NGs from the CoOOH. 4080

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Analytical Chemistry Selectivity of the Nanoprobe. Selectivity is a key factor to evaluate the sensing property of a nanoprobe, especially for the nanoprobe with potential applications in practical biological samples, because the physiological environment is complex, some reactive species such as metal ions and biomolecule would influence the activity of AA. In order to investigate the specificity of the nanosystem for AA, the response of CoOOHNGs to different interfering species containing Na+, Mg2+, Zn2+, Ca2+ Fe2+, Fe3+, Co2+, Ni2+, Mn2+, K+, Cu2+, L-arginine (L-Arg), L-cysteine (L-Cys), L- tyrosine (L-Tyr), glutamic acid (Glu), HPO42−, HCO3−, H3BO4, H2O2, GSH, ClO− and AA was assessed. As shown in Figure 3, compared with other interfering

Figure 4. MTS assay of the CoOOH-NGs with different concentrations.

MTS assay suggested that the nanoprobe exhibits low cytotoxicity and good biocompatibility in living systems. Fluorescence Imaging of AA in Cells. The applicability for TP images of AA in living cells was investigated. In the typical assay, the nanoprobe was incubated with Hela cells for 2.5 h, then the sample was excitated by a femtosecond laser (excitation wavelength at 810 nm) and collected the fluorescence signal in the range of 580−680 nm. Specifically, a 60× water lens was used in the imaging experiments. As shown in Figure 5A, TP fluorescence imaging experiments of AA in living cells were carried out. The incubation of the Hela cells with the CoOOH-modified nanoprobe affords an obvious red fluorescence emission upon excitation by 810 nm femtosecond pulses (Figure 5A,a), which might be caused by the endogenously produced AA of the living Hela cells. In order to confirm our hypothesis, the Hela cells were first incubated with ascorbate oxidase (AOase) (10 U/μL) which could inhibit the activity of endogenous AA. As shown in Figure 5A,b, a very weak red fluorescence was observed in the Hela cells, suggesting that the fluorescence enhancement is indeed triggered by endogenously generated AA and our nanoprobe is suitable for detecting endogenously produced AA of the living Hela cells. To further prove that the fluorescence was correlated with the AA concentration, Hela cells were first incubated with 200 μg/mL CoOOH-NGs for 2.5 h, and then a different concentration of AA (50 μM, 100 μM) was added, with results shown in Figure 5A,c,d. The result indicated that with the AA concentration increased, the fluorescence of the incubation of the Hela cells with the CoOOH-modified nanoprobe was enhanced. We further analyzed the pixel signal intensity of Hela cells imaging by Olympus software to confirm the fluorescence signal intensity was correlation with the AA concentration. The pixel signal intensity of four cells was calculated, and the result was shown in Figure 5B. The fluorescence intensity was increased with the increase of AA concentration. All these results demonstrate that the CoOOH-NGs is suitable for TP imaging of exogenous AA in biological samples. A remarkable advantage of our nanoprobe is its activatable TP fluorescence imaging, which could dwindle the background of cellular autofluorescence. In order to confirm this characteristic, the one-photon and TP fluorescence imaging of AA in cells was performed. As shown in Figure S15, fluorescence image of cells excited by one-photon showed weak fluorescence

Figure 3. Selectivity of the CoOOH-NGs nanoprobes (1 mg/mL) for AA over other electrolytes and biomolecules (AA at concentration of 800 μM and other agents at concentration of 1 mM). (a) NaCl, (b) MgCl2, (c) ZnCl2, (d) CaCl2, (e) FeSO4, (f) FeCl3, (g) CoCl2, (h) Ni(NO3)2, (i) MnCl2, (j) KCl, (k) CuCl2, (l) L-Arg, (m) L-Cys, (n) LTyr, (o) Glu, (p) Na2HPO4, (q) NaHCO3, (r) H3BO4, (s) H2O2, (t) GSH, (u) NaClO, and (v) AA.

molecule, the fluorescence intensity of the nanoprobe has a greater enhancement ratio toward AA. Remarkably, reducing substance such as GSH did not generate apparent interference. Therefore, the CoOOH-modified nanoprobe exhibited better selectivity toward AA sensing and could be applied to detecting AA in living cells and tissue. Detection of AA in Cell Extracts. In order to assess the practicality of the CoOOH-NGs nanoprobe, we performed the analysis of endogenous AA in cell extracts. To eliminate the interference from reducing substance like Hcy, GSH, and Cys, NEM (0.1 mM) was added into the cell extracts. As shown in the Figure S14, with 20-fold diluted cell extracts was added, the fluorescence intensity at 660 nm of the CoOOH-NGs nanoprobe was increased. This results confirmed that the CoOOH-NGs nanoprobe was feasible to detect AA in real biological samples. MTS Assay. The cytotoxicity of the CoOOH-NGs nanoprobe was evaluated by performing MTS assay in Hela cells with different nanoprobe concentrations (20, 40, 60, 80, 100, 200 μg/mL). The cell viability is indirectly reflected by the absorption value at 490 nm of MTS. We chose the cells only incubated with culture medium as blank sample and the ratio of the cells incubated with the nanoprobe to the blank sample was used to assess the cell viability. As shown in Figure 4, it was found that the cell viability of Hela cells was above 90%, with the concentration of the nanoprobe up to 200 μg/mL. The 4081

DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084

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Analytical Chemistry

Figure 5. (A) TP confocal fluorescence microscopy images of Hela cells treated with (a) 200 μg/mL CoOOH-NGs, (b) 200 μg/mL CoOOH-NGs and 10 U/μL AOase, (c) 200 μg/mL CoOOH-NGs and 50 μM AA, and (d) 200 μg/mL CoOOH-NGs and 100 μM AA. (B) Relative pixel fluorescence intensity of the Hela cells images. (a) 200 μg/mL CoOOH-NGs, (b) 200 μg/mL CoOOH-NGs and 10 U/μL AOase, (c) 200 μg/mL CoOOH-NGs and 50 μM AA, and (d) 200 μg/mL CoOOH-NGs and 100 μM AA. TP images were collected at 580−680 nm. Scale bar = 20 μm.

Figure 6. Depth fluorescence images of CoOOH-NGs (200 μg/mL) (A) and CQDs (1 mg/mL) (B) in tissue. The change of fluorescence intensity with scan depth were determined by spectral confocal multiphoton microscopy in the z-scan mode (step size, 5 μm). TP images were collected at 580−680 nm and 400−500 nm, respectively. Scale bar = 100 μm.

signal of NGs while fluorescence image of cells excited by TP showed obvious fluorescence signal, suggesting that TP images could offer superior fluorescence imaging in cells.

TP Imaging of AA in Tissue. The applicability for TP imaging of AA in tissue was investigated by incubating rat liver slice with the nanoprobe. As shown in Figure S16a, there are 4082

DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084

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Analytical Chemistry negligibly weak fluorescence was observed in the rat liver frozen slice incubated with the nanoprobe only. However, strong red fluorescence was observed in the rat liver frozen slice which was treated with the nanoprobe (200 μg/mL) and 200 μM AA (Figure S16b), suggesting the detection and imaging of AA in living tissues is feasible. To further assess the performance of the nanoprobe with NIR emission wavelength for better tissue imaging capacity, the synthesized nanoprobe was prepared with ordinary carbon quantum dots (CQDs) with its emission wavelength at the range of visible light. As shown in Figure 6A, by collecting fluorescence signal in the range of 580−680 nm upon excitation of 810 nm, the CoOOH-NGs is able to make tissue images at depths of 0−320 μm. The tissue imaging results of the CQDs was shown in Figure 6B, by collecting fluorescence signal in the range of 400−500 nm upon excitation of 750 nm the CQDs was able to make tissue images at depths of 0−180 μm. These results demonstrated that the CoOOH-NGs nanoprobe has excellent tissue penetration ability and has better imaging of AA in tissue.

J1210040, 21605091, and 21177036), the National Key Basic Research Program of China (Grant 2013CB932702), National Instrumentation Program (Grant 2011YQ030124), the Foundation for Innovative Research Groups of NSFC (Grant 21521063), Project funded by China Postdoctoral Science Foundation (Grants 2016M592419), Natural Science Foundation of Shandong Province of China (Grants 2016ZRB01098), and the Science and Technology Project of Hunan Province (Grants 2016RS2009, 2016WK2002).

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CONCLUSIONS In summary, we have developed a novel NIR nanoprobe (CoOOH-NGs) with TP excitation for detection and imaging of endogenous AA in living cells and deep tissue imaging. In this system, CoOOH nanoflakes formed on the surface of the NGs could efficiently quench the fluorescence of the NGs by ET from NGs to CoOOH nanoflakes. However, AA could reduce CoOOH to Co2+, so the introduction of AA could reverse the quenching effect induced by CoOOH. Thus, CoOOH-NGs showed the highly sensitive and selective detection of AA. Furthermore, by taking advantage of both TP and NIR fluorescence features such as lower fluorescence background, the nanoprobe was successfully used for the imaging of endogenous AA in living cells and deep tissue imaging. We expect the proposed nanoprobe could supply a novel platform for the analysis of the biological targets in vitro and in vivo.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.6b04943. Supplementary spectral data (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. ORCID

Xiao-Bing Zhang: 0000-0002-4010-0028 Weihong Tan: 0000-0002-8066-1524 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the National Key Scientific Program of China (Grant 2011CB911000), National Natural Science Foundation of China (Grants 21325520, 21327009, 4083

DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084

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DOI: 10.1021/acs.analchem.6b04943 Anal. Chem. 2017, 89, 4077−4084