FRET-Based Upconversion Nanoprobe Sensitized by Nd3+ for the

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FRET Based Upconversion Nanoprobe Sensitized by Nd3+ for the Ratiometric Detection of Hydrogen Peroxide in vivo Han Wang, Yongkuan Li, Man Yang, Peng Wang, and Yueqing Gu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21549 • Publication Date (Web): 23 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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ACS Applied Materials & Interfaces

FRET Based Upconversion Nanoprobe Sensitized by Nd3+ for the Ratiometric Detection of Hydrogen Peroxide in vivo Han Wang, Yongkuan Li, Man Yang, Peng Wang* and Yueqing Gu* State Key Laboratory of Natural Medicines, Department of Biomedical Engineering, School of Engineering, China Pharmaceutical University, Nanjing 210009, China. KEYWORDS: FRET, 808 nm light, ratiometric, hydrogen peroxide, upconversion nanoprobe

ABSTRACT The exorbitant level of hydrogen peroxide is closely related to many human diseases. The development of novel probes for H2O2 detection will be beneficial to disease diagnosis. In this study, a novel Nd3+-sensitized upconversion nanoprobe based on FRET (Förster Resonance Energy Transfer) was firstly developed for sensing H2O2. This nanosystem was made of core-shell upconversion nanoparticles (emission at 540 nm and 660 nm), DCM (dicyanomethylene-4H-pyran)-H2O2 and PAA-octylamine. Obviously, UCNPs doped with Nd3+ acted as an energy donor, and DCM-H2O2, transferring to DCM-OH with the reaction of H2O2, acted as an energy acceptor. The ratiometric upconversion luminescence (540 nm/660 nm) signal could be utilized to visualize the H2O2 level, and the LOD of nanoprobe for H2O2 was quantified to be 0.168 µM. Meanwhile, owing to the dope of Nd3+, the nanoprobe would not induce the overheating effect in biological samples and could possess deeper tissue penetration depth,

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comparing with the UCNPs excited by 980 nm light during bio-imaging. The nanoprobe could also play an important role in detecting the exogenous and endogenous H2O2 in living cells with a ratiometric UCL (upconversion luminescence) imaging. Furthermore, our nanoprobe could function in detecting the H2O2 in a tumor-bearing mouse model. Therefore, this novel nanoprobe with the ratiometric method for responding and bioimaging H2O2 could serve a new technique to furtherance the emerge of novel probes for H2O2 detection.

1. INTRODUCTION Hydrogen peroxide as a significant molecule in signal transduction1 was first discovered by L.J.Thenard in 1818. As one member of the reactive oxygen species (ROS) family, it features in a number of physiological processes.2-6 However, the exorbitant level hydrogen peroxide would induce DNA damage, aging, nonreversible cellular damage, and even cell death.7-10 Therefore, it was established that a high-level hydrogen peroxide was related to a variety of serious diseases like Alzheimer’s disease, malignant tumor, Parkinson’s disease, and inflammation.9, 11-14 Hence, the detection and visualization of hydrogen peroxide in vivo could offer potent evidences in the clinic to promote the diagnosis and monitoring of all these diseases. Fluorescence probes were widely used for the detection and imaging of hydrogen peroxide due to their high sensitivity and specificity. Thus, various fluorescence probes were developed for detecting hydrogen peroxide, which was based on fluorescence changes in the presence of hydrogen peroxide.15-19 Nevertheless, most fluorescence probes were excited by ultraviolet (UV) or visible light when applied to detect hydrogen peroxide in biological samples. Unfortunately, there are several disadvantages of the fluorescence probes excited by UV-vis light, including high background signal, poor tissue penetration, and optical damage to biological samples. To


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solve this problem, the near-infrared (NIR) excited luminescent chemodosimeters were employed for the hydrogen peroxide detection.20-22 However, a majority of NIR-excited sensors suffered from a short Stokes shift and susceptible photobleaching, which have adverse influences on the detecting processes. Different from NIR-excited luminescent chemodosimeters, upconversion nanoparticles (UCNPs) doped by lanthanide possess the superiorities including the anti-Stokes shift of large range, excellent photostability even under longtime irradiation and no autofluorescence in biological samples.23-28 Therefore, the upconversion nanoparticles excited by NIR light have been extensively used in biological detection.29-38 Because of their alterable emission spectra, the energy could be transferred from UCNPs (energy donor) to the luminescent chemodosimeters (energy acceptors), which is called upconversion Förster resonance energy transfer (FRET).39,40 And based on this theory, numerous kinds of nanosystem consisted of UCNPs and luminescent chemodosimeters have been developed to detect different types of samples including ions,41-48 macromolecules,50-52 DNA,53-54 and even proteins.55-56 However, most of the upconversion nanoparticles are doped with Yb3+, that means they must be excited under 980 nm light irradiation during the detecting process. And there is an intense absorption of water to 980 nm light, which could induce the biological samples to generate overheating reaction, leading to the undesired cell death and tissue damage.30, 57-58 To eliminate this adverse effect, Nd3+ serves as a substitute of Yb3+ in UCNPs, which makes the excitation wavelength of UCNPs change to 808 nm, accompanying lower overheating reaction and deeper penetration depth of tissue.59 More importantly, the emission location of UCNPs doped with Nd3+ is in accord with that doped with Yb3+.60 However, the detecting systems based on UCNPs which excited under 808 nm light irradiation are rarely reported.


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Herein, an upconversion nanosystem based on Förster resonance energy transfer (FRET) was first designed to detect hydrogen peroxide with less overheating effect. This nanosystem was made up of NaYF4: 18%Yb, 0.5%Nd, 1%Er@NaYF4: 20%Nd (UCNPs), DCM-H2O2 and PAAoctylamine. Obviously, UCNPs doped with Nd3+ acted as an energy donor, and DCM-H2O2, transferring to DCM-OH with the reaction of hydrogen peroxide, acted as an energy acceptor. PAA-octylamine, acting as amphiphilic micelles, could render UCNPs hydrophilic and adsorb DCM-H2O2. Then, this nanoprobe could sense hydrogen peroxide in the living tumor cells by using ratiometric upconversion luminescence (540 nm/660 nm) imaging, and it also could probe the hydrogen peroxide in the tumor-bearing mice model. 2. EXPERIMENTAL SECTION 2.1. Materials and Reagents. 2-(2-Methyl-4H-chromen-4-ylidene)malononitrile,4-(4,4,5,5tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde, RE(CH3COO)3 (Y, Yb, Nd and Er), oleic acid, 1-octadecene, n-octylamine, poly(acrylic acid) (PAA, Mw 3000, 50%), N-(3Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDCI) and N-Hydroxysuccinimide (NHS) were obtained from J&K scientific; lipopolysaccharide (LPS) and 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Aladin Reagent Co; NH4F, NaOH, ethanol, N,N-dimethylformamide (DMF), piperidine, cyclohexane, dimethylsulfoxide (DMSO), chloroform, methanol, H2O2, and acetic acid were obtained from Sinopharm chemical reagent Co; fetal bovine serum, Trypsin Dulbecco’s Modified Eagle’s Medium (DMEM) were obtained from Thermo Fisher Scientific. Penicillin, streptomycin, phosphate buffered saline (PBS), were purchased from Aladin Reagent Co. And human hepatocellular carcinoma cell line (HepG2) was purchased from KeyGen Biotech (Nanjing, China); athymic nude mice (female, 25 g, 5–7 weeks old) were obtained from Laboratory Animal Resources of China Pharmaceutical


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University. And all animal operation procedures were approved according to the Animal Care Guidelines of China Pharmaceutical University. 2.2. Synthesis of DCM-H2O2 2-(2-Methyl-4H-chromen-4-ylidene) malononitrile (1.0 mmol) and 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzaldehyde (1.0 mmol) were added in 10 mL anhydrous ethyl alcohol and stirred until the mixtures were dissolved. Then piperidine (4.0 mmol) was added into the system and heated to reflux for 5 h. At last, the reaction mixture was filtered to afford DCM-H2O2 as yellow solid (yield 77%) (Figure S1). 1H-NMR (300 MHz, CDCl3): δ 8.93 (d, J = 8.2 Hz, 1H), 7.88 (d, J = 7.8 Hz, 2H), 7.75 (t, J = 7.8 Hz, 1H), 7.62 (m, 4H), 7.46 (t, J = 7.7 Hz, 1H), 6.90 (t, J = 8.0 Hz, 2H), 1.36 (s, 12H) (Figure S2). 2.3.

Synthesis

of

NaYF4:Yb/Nd/Er@NaYF4:Nd(20%)

Core-shell

Upconversion

Nanoparticles. The synthetic method of NaYF4:Yb/Er/Nd was according to a research of Xie before.59 6 mL oleic acid and 15 mL 1-octadecene were transferred into four-neck-flask, then 1 mM RE(CH3COO)3 containing Y, Yb, Er, Nd (ratio=80.5%:18%:0.5%:1%) were added into the mixture. After the air extraction by using nitrogen gas, the mixed solution was heated at 150 oC for 1 h, the temperature of mixed solution was decreased to 50 oC subsequently. Whereafter, 5 mL methanol including NaOH (2.5 mM) and NH4F (4 mM) was transferred into mixed solution. 30 min stirring later, the temperature of the mixture was raised at 100 oC to eliminate methanol. After 30 min heating, the mixed solution was heated to 290 oC. And the temperature of mixture was cooled to 20 oC after 1.5 h calefaction. The mixture was sublimated three times with cyclohexane and anhydrous ethanol. As to the synthesis of NaYF4:Yb/Nd/Er@NaYF4:Nd(20%), following the above procedure, when the temperature of mixed solution was dropped to 50 oC. 6 mL above UCNPs and 6 mL


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methanol including NaOH (2.5 mM) and NH4F (4 mM) was transferred into the mixture for 30 min stirring. After evaporating methanol and cyclohexane, the mixed solution was heated to 290 oC.

And the temperature of mixture was cooled to 20 oC after 1.5 h calefaction. The mixture was

sublimated three times with cyclohexane and anhydrous ethanol. 2.4. Synthesis of PAA (Poly Acrylic Acid)-n-octylamine (PAAO). The synthesis process of PAAO was followed as the procedure published before.30 Simply, 0.48 g NHS and 0.8 g EDCI were transferred into the 3 mL DMF containing 0.6 g PAA. After 6 h stir, 233 µL n-octylamine in 1 mL DMF was transferred into the mixed solution. After 12 h stir, the mixed solution was purified by using dialyzing for three days. 2.5. Assembly of PAAO-UCNPs-DCM-H2O2.The assembly of PAAO-UCNPs-DCM-H2O2 was followed as the procedure published before.30 500 µL containing 20 mg UCNPs was added into 5 mL PAAO solution (15 mg), then the mixture was transferred into ultrasonic cell disruptor with 20% intensity for 5 min. Then, 500 µL DMSO containing 0.5 mg DCM-H2O2 was added into the mixture. After 12 h stiring, the final mixture was purified with 10 min centrifugation at 3000 rpm. Finally, the DCM-H2O2 loading efficiency in PAAO was calculated by using the supernatant. The absorption of DCM-H2O2 with different concentrations at 415 nm and 435 nm was presented in Figure S6 and S7. After calculating, 0.352 mg DCM-H2O2 was loaded into 15 mg PAAO. In addition, the efficiency of FRET between UCNPs and DCM-H2O2 by the following equation: E=1-UCL/UCL0 (E: efficiency of FRET, UCL: integrated emission intensities of UCL530nm-560nm with addition of H2O2, UCL0: integrated emission intensities of UCL530nm-560nm without addition of H2O2)


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2.6. Cell culture. HepG2 cells were cultured with DMEM medium in 37 oC and 5% CO2 incubator, supplemented with 10% FBS, penicillin(100 U mL-1), and streptomycin(100 U mL-1). 2.7. Cell viability of nanoprobe The cell viability of nanoprobe was measured by using methyl thiazolyl tetrazolium (MTT) assay. Briefly, HepG2 cells in logarithmic phase were cultured in a 96-wells plate with 1*104 cells per well. After incubating overnight, the nanoprobe was added into the plate with concentrations of 100, 200, 300, 400 and 500 µg mL-1 for 24 h and 48 h incubation at a 37 oC and 5% CO2 incubator, respectively. Then, the MTT (20 μL; 5 mg mL-1) solution was added into the medium with another 4 h incubation. At last, the medium in the plate was removed and 150 μL DMSO was added into the per well. The absorption at 495 nm of per well was measured by a microplate reader. 2.8. Upconversion luminescence imaging of PAAO-UCNPs-DCM-H2O2 in vitro. The HepG2 cells (5 *104 per dish) were inoculated in confocal dishes. After incubating overnight in a 37 oC and 5% CO2 incubator, 200 μg mL-1 nanoprobe was transferred into the confocal dish for incubating 8 h. Then, the medium was replaced with fresh medium including 10 μM H2O2 solution. After incubating for 2 h, the green and red UCL images were obtained by using laser scanning confocal microscope excited by 808 nm light. The green (500–560 nm) and red (600– 700 nm) UCL emission signals were collected, respectively. As to the response of endogenous H2O2 by nanoprobe, The HepG2 cells (5 *104 per dish) were inoculated in confocal dishes. After incubating overnight at a 37 oC and 5% CO2 incubator, 20 μM LPS solution was added into the medium to induce H2O2 in HepG2 cells. After 12 h incubation, the medium was replaced with fresh medium including 200 μg mL-1 nanoprobe. After incubating for 8 h, the green and red UCL images were obtained by using laser scanning


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confocal microscope excited by 808 nm light. The green (500–560 nm) and red (600–700 nm) UCL emission signals were collected, respectively. 2.9. Upconversion luminescence imaging of PAAO-UCNPs-DCM-H2O2 in vivo. HepG2 cells (5*105) were subcutaneously injected left axilla of the 5 weeks-old nude mice (n=5). When the tumor volume was 110 mm3, 50 µL PAAO-UCMPs-DCM-H2O2 was injected into the tumor. Meanwhile, the same dosage PAAO-UCMPs-DCM-H2O2 was subcutaneously injected right axilla of the same mouse. Then, the images of the green UCL of the tumor-bearing nude mouse was taken by a NIR imaging system under 808 nm laser irradiation with a power density of 400 mW/cm2. The assay of fluorescence intensity was investigated by image J with a semi quantitative method. 3. RESULT AND DISCUSSION 3.1. Design of upconversion nanoprobe for hydrogen peroxide detection. In this research, the detection system for H2O2 was based on Förster resonance energy transfer (FRET). Therefore, the emission spectrum of energy donor must overlap with the absorption spectrum of energy acceptor. The UCNPs excited by 808 nm light could serve as an energy donor, while DCM-H2O2 could serve as an energy acceptor. To achieve the required distance of FRET and improve the water solubility of UCNPs and DCM-H2O2, PAAO and amphiphilic micelles were applied into the nanosystem. (Scheme 1) Due to the doping of Er3+, the energy could transfer from Nd3+ to Yb3+ to Er3+. Then UCNPs possessed two emission peaks under 808 nm light irradiation, which mainly located at 540 nm and 660 nm. The absorption of DCM-H2O2 with donor–-acceptor (D––A) structure was around 440 nm, and there is no FRET between UCNPs and DCM-H2O2. Once H2O2 was added in this nanosystem, the boronic ester group of DCMH2O2 was transformed to phenolic hydroxyl with change of ICT (intramolecular charge transfer),


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which was named as DCM-OH (Figure S3). The absorption of DCM-OH was located at ~559 nm, which perfectly overlapped with the emission of UCNPs of the 2H11/2 → 4I15/2, 4S3/2 → 4I15/2 transition, and the green UCL (540 nm) of UCNPs was quenched. The emission change of nanosystem could serve as a detection signal along with the addition of H2O2. Scheme1. Schematic illustration of the synthesis and response process for H2O2 of upconversion nanoprobe

3.2. Optical Response of DCM-H2O2 to H2O2. As an energy acceptor in the nanosystem, the reaction activity of DCM-H2O2 to H2O2 was assessed in a PBS-DMSO (1:1, v:v) solution. In the presence of H2O2, the boronate group of DCM-H2O2 could transform into the phenolic hydroxyl group to afford DCM-OH successfully. As shown in Figure 1, the absorption and emission spectra were obtained after adding different concentrations of H2O2 solution. With the addition of H2O2, the absorption peak of DCM-OH at ~559 nm gradually increased, and the color of the


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solution changed from yellow to brown, implying that DCM-H2O2 could react with H2O2. (Figure 1A) Similarly, the fluorescence emission of DCM-OH at ~695 nm enhanced linearly. (Figure 1B) These results suggested that DCM-H2O2 could serve as a candidate sensor to detect H2O2. Unfortunately, due to the overlap between the absorption spectra (520 nm-650 nm) and emission spectra (600 nm-780 nm), the detection process was susceptible to the excitation light. But this issue could be overcome by establishing an upconversion nanosystem.

Figure 1. (A) The absorption spectra of DCM-H2O2 (20 µM) after adding different concentrations H2O2 solution. (B) The fluorescence emission spectra of DCM-H2O2 (20 µM) with incremental addition of H2O2 solution (0, 0.5, 1, 2, 4, 6, 8, and 10 µM). 3.3. Synthesis and Characterization of nanoprobe. To establish the nanosystem, UNCPs (NaYF4: Yb, Er, Nd) coated by oleic acid were firstly synthesized according to previous methods. Due to the doping of Nd3+, the UCNPs (NaYF4:Yb, Er, Nd) could be excited under 808 nm light irradiation. To improve the fluorescence intensity of UCNPs (NaYF4: Yb, Er, Nd), the core-shell UCNPs (NaYF4: Yb, Nd, Er@NaYF4, Nd) was synthesized based on NaYF4:Yb, Er, Nd. The TEM (transmission electron microscopy) images were employed to characterize the morphology and size of UCNPs. Figure 2A showed that the size of NaYF4:Yb, Er, Nd was ~21.1


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nm. After the formation of the core-shell structure, the size of NaYF4: Yb, Nd, Er@NaYF4, Nd has increased to ~23.6 nm (Figure 2B). Furthermore, the XRD patterns presented in Figure 2D showed that the X-ray diffraction peaks of UCNPs had a ideal match with the standard pattern of pure hexagonal NaYF4 (JCPDS No.28-1192). Moreover, the UCNPs was hydrophobic after the modification of oleic acid on the surface of UCNPs (NaYF4: Yb, Nd, Er@NaYF4, Nd). Therefore, amphipathic micelle PAAO was employed to render UCNPs hydrophilic by hydrophobic interaction. Then DCM-H2O2 was adsorbed into the PAAO by the similar principle. And Figure 2C revealed that the TEM image of the nanosystem in deionized water was ~26.3 nm. In addition, the particle size of nanosystem in deionized water measured by dynamic light scattering was 74.3 nm (Figure 2E). The dispersity (PdI) of nanosystem was 0.188, implying that it possessed a good dispersity. The photobleaching experiment showed that both the green and red UCL of UCNPs almost maintained their original value after the continuous 808 nm light irradiation for 120 min, implying that the nanoprobe possessed high resistance to photobleaching (Figure S8). At last, the stability of nanoprobe for different pH was assessed. The emission spectra of nanoprobe under different pH values (pH=4, 5, 6 and 7) was presented in Figure S9. And the different pH environments almost did not affect the fluorescent intensities of nanoprobe under 808 nm light irradiation. The absorption spectra of DCM-H2O2, PAAO, and PAAO-UCNPs-DCM-H2O2 were shown in Figure S4. DCM-H2O2 exhibited the absorption peaks at ~420 nm and ~440 nm, and the absorption of nanosystem was in accord with DCM-H2O2, indicating that DCM-H2O2 was introduced into PAAO-UCNPs successfully. To further confirm the assembly of PAAO and DCM-H2O2 on the UNCPs, the characteristic absorption of UCNPs, PAAO-UCNPs, DCM-H2O2, and nanosystem were evaluated by Fourier-transform infrared (FTIR) spectroscopy. As shown in


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Figure S5, the absorption peaks at 2924.5 cm-1 and 2853.5 cm-1 in the FTIR spectrum of UCNPs were attributable to the stretching vibrations of methylene (-CH2-), and the absorption peak located at 1564.8 cm-1 belonged to the vibration of carbonyl (C=O). Moreover, the absorption peaks of 3412.3 cm-1 and 1409.9 cm-1 were attributable to the acid amides bond (C-N) in the spectrum of PAAO-UCNPs. The absorption of 1649.1 cm-1 was assigned to the vibration of the carbon-carbon double bond (C=C) in the spectrum of DCM-H2O2. The absorption peaks above were contained in the spectrum of nanosystem, which suggested that PAAO and DCM-H2O2 were assembled on the UCNPs favorably. The emission spectrum of UCNPs and the absorption spectrum of DCM-OH were illustrated in Figure 2F. The absorption of DCM-OH (520 nm-650 nm) overlapped well with the emission of UCNPs (515 nm-560 nm) under the excitation at 808 nm light, indicating that the FRET process could proceed smoothly between UCNPs and DCM-OH. More importantly, the absorption of DCM-OH hardly overlapped with the red emission of UCNPs (640 nm-675 nm) and had nearly no influence on the red emission intensity, implying it could be served as a candidate of reference in the ratiometric response of H2O2.


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Figure 2 (A) The TEM image of UCNPs (NaYF4:Yb, Er, Nd). (B) The TEM image of core-shell UCNPs (NaYF4: Yb, Nd, Er@NaYF4, Nd). (C) The TEM image of PAAO-UCNPs-DCM-H2O2. (D) X-ray diffraction pattern of the core-shell UCNPs and the standard pattern of β-NaYF4. (E) The particle size of nanoprobe in deionized water. (F) the absorption spectrum of DCM-OH and the emission spectrum of UCNPs. 3.4.The sensitive and selective detection of H2O2 by nanoprobe. The sensing ability of nanoprobe for H2O2 was assessed with the application of UV-vis absorption and the UCL emission spectra. As shown in Figure 3A, the absorption peaks of nanoprobe were located at ~559 nm after adding various concentrations H2O2 solutions. And the absorbance of nanoprobe increased linearly after adding a series of H2O2 solutions (0-10 µM) , indicating that the DCMH2O2 inside of the nanoprobe could react with H2O2. (Figure 3A) Meanwhile, the FRET process between UCNPs and DCM-OH would be influenced because of the absorption strength changed of DCM-OH. Figure 3B displayed the green UCL emission of nanoprobe with the addition of H2O2 solutions in the range of 0-10 µM. And the green UCL intensities (515 nm-560 nm) of


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nanoprobe under 808 nm laser irradiation decreased gradually with the increase of the H2O2 concentrations. That was ascribed to the increase FRET effect between UCNPs and DCM-OH. Namely, the increase absorbance of DCM-OH (~559 nm) had more overlap with the green UCL of UCNPs as the increase of H2O2 concentrations. Therefore, the ratio of the intensity at 540 nm over 660 nm could be served as a detection signal for H2O2. And the efficiency of FRET between UCNPs and DCM-OH is 88.15% after adding 10 µM H2O2 solution (Figure 3B). Owing to the application of the ratiometric method in the detection process of H2O2, the interference of concentrations and other factors could be avoided commendably. Furthermore, as a detection nanoprobe for H2O2, the detectability was important. The LOD of nanoprobe for H2O2 was 0.168 µM by using ratiometric method excited by the 808 nm laser, implying that it could be used in biological detection for H2O2 due to its high sensitivity and low background signal.

Figure 3 (A) The absorption spectra of nanoprobe with incremental addition of H2O2 solution (0, 0.5, 1, 2, 4, 6, 8, and 10 µM). (B) The emission spectra of nanoprobe with incremental addition of H2O2 solution (0, 0.5, 1, 2, 4, 6, 8, and 10 µM) excited by 808 nm light. As a detection nanoprobe for H2O2, high selectivity is significant. And the detection specificity of nanoprobe was investigated by responding with a variety of reactive oxygen


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species (ROS) and relevant nitrogen species (RNS) solutions under the same condition. As presented in Figure 4A, RNS and ROS solutions (ClO-, ONOO-, 1O2, O2-, .OH, NO, and H2O2) were added into the nanoprobe respectively, and only the addition of H2O2 solution made the color of nanoprobe change brown. The absorption spectra of nanoprobe reacting with RNS and ROS were measured by ultraviolet spectrophotometer. Figure 4B showed that only the nanoprobe solution possessed a new absorption located at ~559 nm. However, the absorbances of the other groups did not change. Then, the emissions of nanoprobes were obtained after adding the RNS and ROS solutions (Figure 4C). And the green UCL emission of nanoprobe decreased substantially with the addition of H2O2 solution, while the green UCL emissions in other groups remained invariant. At last, the typical biomolecules (glucose, glutathione, dopamine and ascorbic acid), ions (Na+, K+, Mg2+, Ca2+, Fe3+, Cl-, F-,CO32- and PO43-) and amino acids (valine, glycine, cysteine, glutamic acid and lysine) were chosen for the selectivity assay of nanoprobe, and the similar results were obtained (Figure S10, 11,and 12). These results above indicated that the nanoprobe could respond with H2O2 selectively among RNS and ROS solutions.


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Figure 4 (A) The color changes of nanoprobe by adding different ROS and RNS solution. (B) The absorption spectra of nanoprobe after responding with different ROS and RNS solution. (C) The emission spectra of nanoprobe after responding with different ROS and RNS solution. (D) The ratio between UCL540

nm

and UCL660

nm

after responding with different ROS and RNS

solution. 3.5. Cytotoxicity of PAAO-UCNPs-DCM-H2O2. As a detection system applied in the biological field, the nanoprobe needs to be innoxious and safe to the cells and tissue. And the toxicity of nanoprobe was investigated by using the MTT assay. HepG2 cells were chosen as a cell model in the process. As shown in Figure 5, after adding the nanoprobe with different concentrations (0 µg/mL, 100 µg/mL, 200 µg/mL, 300 µg/mL, 400 µg/mL, 500 µg/mL), the cell viabilities of HepG2 cells were not affected. Even if the incubation time was extended to 48 h, the cell viability of the group with the highest nanoprobe concentration was 88%. The results


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suggested that the nanoprobe possessed low toxicity, and it could be used for the detection in the biological field.

Figure 5 The cell viability of HepG2 cells with incremental addition of PAAO-UCNPs-DCMH2O2 by incubating 24 h and 48 h. 3.6.Responding H2O2 in living cells. To evaluate the usability of nanoprobe for detecting H2O2 in living cells, the UCL images of nanoprobe were obtained by using confocal scanning laser microscopy. First, the distribution of the nanoprobe in HepG2 cells was assessed, and most of the green UCL of nanoprobe co-localized with endosome after the Lyso-Tracker dye incubation. Nevertheless, the other nanoprobe distributed in the cytoplasm, which could probe H2O2 in the cytoplasm (Figure S13). Then, the UCL images in HepG2 cells for H2O2 detection were taken after adding the nanoprobe for 8 h subsequently. There was a strong green UCL and red UCL of nanoprobe in Figure 6B and 6C. Once the HepG2 cells in confocal dishes were added H2O2 for 2 h incubation under the same condition, the green UCL reduced obviously (Figure 6G), which implied that the nanoprobe could detect the intracellular H2O2. Moreover, the ratiometric UCL imaging (ratio=UCL540nm/UCL660nm) showed that the UCL ratio by added


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nanoprobe in HepG2 cells was >0.75 (Figure 6E). However, the HepG2 cells after adding H2O2 under the same condition presented a UCL ratio of 0.9 (Figure 7E), and the UCL ratio by incubating LPS in


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the HepG2 cells under the same condition was

FRET-Based Upconversion Nanoprobe Sensitized by Nd3+ for the

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