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In Vivo Oxidative Stress Monitoring Through Intracellular Hydroxyl Radicals Detection by Recyclable Upconversion Nanoprobes Yuxin Liu, Qi Jia, Quanwei Guo, Anqi Jiang, and Jing Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03270 • Publication Date (Web): 07 Nov 2017 Downloaded from http://pubs.acs.org on November 7, 2017

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Analytical Chemistry

In Vivo Oxidative Stress Monitoring Through Intracellular Hydroxyl Radicals Detection by Recyclable Upconversion Nanoprobes

Yuxin Liu, Qi Jia, Quanwei Guo, Anqi Jiang, Jing Zhou*

Department of Chemistry, Capital Normal University, Beijing 100048, China E-mail: [email protected]; Tel: +86-010-68902491

Abstract Oxidative stress, as an essential cause to many disease via irreversible biomolecule oxidation, can be induced by casual-contacted nanomaterials, which can generate hydroxyl radical (·OH) due to their active surface capacity. Herein, we report a novel upconversion nanoprobe for ratiometric ·OH detection and monitoring titanium oxide nanomaterial-induced oxidative stress. Unlike previously developed nanoprobes, the ·OH-responsive acceptor in these nanoprobes can be rapidly prepared and easily recycled. With a detection limit of ~2 nM and a broad linear range of 4 nM–16 µM, the nanoprobe is suitable for both in vitro and in vivo applications. The facile preparation and recyclable strategy provide a new method for developing novel nanoprobes that can directly detect hazardous molecules in biological samples and systems. Keywords: upconversion, hydroxyl radicals, oxidative stress, luminescence, recyclable

Introduction Oxidative stress in vivo nearly correlates with the process of forming and developing many diseases, through the irreversible biomolecule oxidative damage, like nucleic acid mutation, lipid peroxidation, and protein degradation.1,2 By generating intracellular hydroxyl radicals (·OH), nanomaterials have been considered as a potential risk factor for inducing oxidative ACS Paragon Plus Environment


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stress in vivo.3 Titanium oxide nanomaterial (nano TiO2), a casual-contacted nanomaterial in our daily life, has broad applications in pigments and photocatalysts due to their relative high surface area and strong photoactivity.4-6 Under ultraviolet irradiation, nano TiO2 can be excited and electrons in the valence band are promoted to the conduction band, which results in electron-hole pair separation and movement from inside to the surface.7 The high oxidative potential of surface holes allows the particles to oxidize water to form ·OH. Therefore, frequently exposure to nano TiO2 presents a serious danger to human health.8 Thus, it is necessary to develop an easy-to-use method to evaluate nano TiO2-induced oxidative stress levels by monitoring the ·OH concentration.9 As the optical signals from chromaticity or luminescence can be easily observed by devices or even the naked eyes, these optical methods have great potential in addressing this issue.10-13 Up to now, a great deal of effort has been made in developing nanoprobes for the highsensitivity optical detection of ·OH by luminescence methods.14-17 Compared with conventional

luminescence

nanoprobes,

near-infrared

(NIR)-excited

upconversion

luminescence nanoprobes (UCNPs) have the advantages of no autofluorescence from biosamples, no photobleaching and deep penetration depth.18-22 Therefore, UCNPs are outstanding candidates for monitoring ·OH concentration in biological samples.23-28 Recently, many groups have highlighted the capability of UCNPs for high-sensitivity ·OH detection.2932

By efficient Förster resonance energy transfer (FRET), UCNPs emission intensity at a

specific wavelength range changes when the acceptor reacts with ·OH.33-35 However, typical acceptors, which are primarily derivatives of organic dyes with azo and triphenylmethane structure, have complex synthetic routine and are potentially carcinogenic, largely limiting their real-world application. Moreover, reactions between the acceptor and ·OH are irreversible and, consequently, these nanoprobes are disposable. Considering the above facts, novel recyclable UCNPs with a facile preparation method are required for the easy-to-use detection of ·OH generated from nano TiO2-induced oxidative stress. ACS Paragon Plus Environment

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Herein, low-toxic recyclable, 4-amino salicylic acid-iron(II) complex (4-ASA-Fe(II))modified NaLuF4:Yb,Er,Tm upconversion nanoparticles are reported for high-sensitivity ratiometric detection of ·OH generated from nano TiO2-induced oxidative stress. The ·OHresponsive acceptor, 4-ASA-Fe(II), can be synthesized via a facile complexation reaction between FeII and phenolic hydroxyl group within seconds. Furthermore, the ·OH can oxidize FeII in complex to FeIII, which results in the formation of 4-ASA-Fe(III) complex and strong absorbance through the d-d transition. The overlay of 4-ASA-Fe(III) complex absorbance and NaLuF4:Yb,Er,Tm emission can lead to the generation of FRET and selectively luminescence quenching. Importantly, this oxidation process is reversible, which suggests that this nanoprobe can be recycled by reductive treatment (Scheme 1). The limit of detection, linear detection range, and selectivity are studied in detail to illustrate the capacity of this nanoprobe for high sensitivity ·OH detection. Moreover, we demonstrate the ratiometric luminescence detection of ·OH generated from nano TiO2-induced oxidative stress both in cells and in a mouse model.

Experimental Section Preparation of recyclable upconversion nanoprobe in reduced state (rUCNP) The NaLuF4:Yb,Er,Tm upconversion nanoparticles were synthesized via a typical solvothermal method.36,37 The oleic acid ligands on the surface of as-prepared nanoparticles were removed by a reported nitrosonium tetrafluoroborate-mediated ligand exchange method.38 Further modification of 4-amino salicylic acid-iron(II) complex was carried out in deionized (DI) water to obtain upconversion nanoprobe in reduced state (rUCNP). Please see Supporting Information for experiment details. Recycle capacity test: 1 mM rUCNP solution was used for recycle study. Hydroxyl radical (·OH) was generated by a reported method and all the involved materials were purchased from Sigma Aldrich.39 ACS Paragon Plus Environment


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After add 1 mM ·OH solution and 20 mM reducing agent (reduced glutathione or ascorbic acid) solution in turn, the nanoprobe was separated via centrifugation and redispersed in DI water for ultraviolet-visible-near infrared (UV-vis-NIR) and upconversion luminescence (UCL) spectra determination. The ratio of luminescence intensity at 800 nm and 540 nm (800 nm/540 nm) were recorded and then calculated to evaluate the recycle capacity. ·OH detection capacity study ·OH solutions with various concentrations of 0-10 mM were added into 1 mM rUCNP solution, incubated for 10 minutes and protected from light. Then, the UV-vis-NIR and UCL spectra of all samples were measured, respectively. Nano TiO2-induced oxidation stress level evaluation The synthesis of nano TiO2 and further modification was taken under the direction of previous reports.41,42 Hepatocyte (CCC-HEL-1) cells were provided by Institute of Basic Medical Sciences Chinese Academy of Medical Sciences and grown in DMEM (Dulbecco’s modified Eagle’s medium) containing 10% FBS (fetal bovine serum) and 1% penicillinstreptomycin at 37 oC with 5% CO2. The experiment of oxidation stress level evaluation in hepatocyte was taken as our previous work states.32 Healthy balb/c nude mice were used as small-animal model for in vivo experiment. All the animal procedures were in agreement with institutional animal use and care committee and carried out ethically and humanely. Mice uptake nano TiO2 by intravenous injection before imaging. Please see Supporting Information for experiment details.

Results and Discussion Characteristic of recyclable upconversion nanoprobes The NaLuF4:Yb,Er,Tm upconversion nanoparticles were controllably prepared via a typical solvothermal method.36,37 Transmission electron microscopy (TEM) images suggested that NaLuF4:Yb,Er,Tm nanoparticles were monodispersed with a regular spherical shape and ACS Paragon Plus Environment

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narrow size distribution of 21.1 ± 1.2 nm, which was in the suitable size range for a Förster resonance energy transfer (FRET) donor according to previous reports (Figure 1A).40 The electron diffraction X-ray analysis (EDXA) pattern and inductively coupled plasmonic-mass spectrum (ICP-MS) results illustrated the chemical composition and elemental ratio of NaLuF4:Yb,Er,Tm nanoparticles (Figure S1, Lu: 82.78%, Yb: 14.97%, Er: 2.13%, and Tm: 0.12%). Moreover, the powder X-ray diffraction (XRD) pattern of NaLuF4:Yb,Er,Tm corresponded to the standard card of the hexagonal phase of NaLuF4 (Figure S2, JCPDS: 0270726). Lattice fringes of (120), (111), and (011) identified from selected-area electron diffraction (SAED) patterns obtained from the Fourier transformation of high-resolution TEM (HR-TEM) images also provided strong evidence of the single-crystalline hexagonal NaLuF4 structure (Figure 1B). Since the 4-ASA-Fe(III) complex had strong absorbance in the range of 400-600 nm, which exactly overlapped with green emission band of NaLuF4:Yb,Er,Tm nanoparticles, 4-ASAFe(II) complex was optimized as the acceptor for ·OH responsive. The 4-ASA-Fe(II) complex was modified via electrostatic forces between positive charges of the NaLuF4:Yb,Er,Tm nanoparticles and the negative carboxyl in the complex (Figure S3), which can be recognized as the reduced state of UCNP (4-ASA-Fe(II) complex modified NaLuF4:Yb,Er,Tm nanoparticles, rUCNP). Fourier transform infrared (FTIR) spectra demonstrated the successful modification of 4-ASA-Fe(II) complex to the NaLuF4:Yb,Er,Tm nanoparticles (Figure 1C). The amount of 4-ASA-Fe(II) complex loading was determined and calculated to be 33.2% (w/w). ICP-MS results suggested no obvious Fe leakage from the rUCNP surface because the strong coordinate covalent bonds between FeII and phenolic hydroxyl group in 4-ASA. After ·OH was added, a strong absorbance in the range of 400–600 nm was observed within 10 s, and the absorbance intensity had a positive relationship with ·OH concentration, which was evidenced by the rapid oxidation of FeII to FeIII (4-ASA-Fe(III) complex modified NaLuF4:Yb,Er,Tm nanoparticles, oUCNP) and can be described as the transition between t2g ACS Paragon Plus Environment


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and eg of 4-ASA-Fe(III) complex (Figure 1D and Figure S4). Moreover, when oUCNP were treated with reducing agents, such as glutathione and ascorbic acid, the absorbance peak disappeared, which indicated the recycling capacity of rUCNP (Figure 1E). It is noteworthy that rUCNP had good stability through the recycling process and can be recycled at least 50 times without observable changes in TEM images, hydrodiameter, ultraviolet-visible-near infrared (UV-vis-NIR), and upconversion luminescence (UCL) spectra (Figure 1F and Figure S5, stipulating 800 nm/540 nm of rUCNP = 0 and oUCNP = 1), which is advantageous compared with previously reported upconversion nanoprobes for ·OH detection (Table 1). Importantly, the absorbance of 4-ASA-Fe(III) complex overlapped with the emission peaks of NaLuF4:Yb,Er,Tm nanoparticles at 540 nm, which correspond to the 2H11/2→4I15/2 and 4

S3/2→4I15/2 transition of Er3+ (Figure 2A). Therefore, FRET generation can occur between

NaLuF4:Yb,Er,Tm nanoparticles and 4-ASA-Fe(III) complex, which leads to the efficient quenching of green emission (540 nm), less influence to red emission (654 nm: 4F9/2→4I15/2 of Er3+), and no obvious effect on NIR emission (800 nm: 3H4→3H6 of Tm3+) (Figure 2B). As a result, a ratiometric detection of ·OH based on rUCNP can be achieved. Ratiometric luminescence method for ·OH detection Luminescence lifetime was first investigated to test FRET generation and calculated the energy transfer efficiency (Figure 3A). The results suggested that rUCNP had a longer luminescence lifetime (582 µs) than oUCNP (349 µs). From these data, the energy transfer efficiency was calculated to be 40.03% (Equal 1 in Supporting Information). This high-energy transfer efficiency ensured the outstanding ·OH response capacity of the nanoprobe. Further efforts were made to study the UCL detection method by determining the UCL spectra of a mixture containing rUCNP (1 mM, pH = 7.4) and various ·OH concentrations (0–1 mM). As shown in Figure 3B, the luminescence intensity at 540 nm decreased accordingly with increasing ·OH concentrations, and luminescence at 654 nm was slightly quenched as well.

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As the nanoprobe has multiple emission peaks that showed different response levels to ·OH concentration, a ratiometric luminescence method can be proposed for further application. To validate this approach, the luminescence intensity ratio between any two wavelengths, centered at 540 nm, 654 nm, and 800 nm, were then calculated, respectively (Figure S6). The results suggested that 800 nm/540 nm was the most sensitive to ·OH concentration changes. Moreover, it can be concluded from the results that the 800 nm/540 nm was well linearly correlated with ·OH concentration (800 nm/540 nm = 1.3818 + 0.2589 ·OH/µM, R2 = 0.994) in the 4 nM–16 µM range (Figure 3C). More importantly, it is noteworthy that the luminescence ratio change would lead to luminescence color variation, which can be identified by the naked eye (Figure 3D). It is also found that rUCNP showed less significant response to other ions, amino acids, biomolecules, and ROS (Figure 3E and Figure S7). Based on the aforementioned results, rUCNP were feasible for detecting the ·OH concentration in biosamples via a luminescence ratiometric method with a low detection limit of 2 nM (signalto-noise > 3) and a broad linear detection range (4 nM–16 µM). Detection of ·OH generated from titanium oxide nanomaterial (nano TiO2)-induced oxidative stress Nano TiO2, a typical exogenous sensitizer, was extensively used in daily life as a pigment and UV absorbent. The wide application of nano TiO2 can lead to an increased risk of oxidative stress-induce toxicity because of frequent exposure. Therefore, nano TiO2 was selected as the ·OH generating and oxidative stress inducing agent in this work. Anatase nano TiO2 was prepared via a modified solvothermal method.41 Basic characteristic showed that nano TiO2 was irregular in shape with a narrow size distribution of 26.98 ± 3.2 nm and pure anatase phase (Figure S8A–C). Bare hydrophilic nano TiO2 was obtained via a reported ligand exchange method.38 After a typical bovine serum albumin (BSA) modification method, the hydrodiameter increased from 32.52 nm to 35.79 nm (Figure S8D), which suggested that an albumin shell of ~1.7 nm was coated onto the surface of bare nano TiO2 (nano TiO2ACS Paragon Plus Environment


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BSA).42 For in vitro studies, the hepatocyte cell line (CCC-HEL-1) was incubated with the obtained nano TiO2 or nano TiO2-BSA and ·OH concentrations at various time points were determined by the rUCNP-based luminescence ratiometric method (Figure 4A). The results suggested that low concentration ·OH was found in nano TiO2-free hepatocytes, however; after incubation with nano TiO2, the ·OH concentration in hepatocytes continuously increased over 7 h, and a much more rapid accumulation was observed under UV irradiation (Figure 4B). We also found that cells incubated with high concentration nano TiO2 produced more ·OH than those with low concentration nano TiO2 (Figure 4C). Moreover, cells incubated with nano TiO2-BSA had significant lower ·OH accumulation than the nano TiO2-incubated cells, which may be attributable to the anti-oxidant capacity of the thiol group in BSA (Figure 4D). These results illustrated that irradiation, nano TiO2 concentration, and surface properties can all influence the production and accumulation of ·OH in hepatocytes. We further illustrated ratiometric ·OH detection through luminescence imaging in vivo (Figure 4E). Since nanomaterials with relatively large hydrodiameters tended to accumulate in reticuloendothelial system, such as the liver and spleen, nano TiO2 had high retention in liver tissue after intravenous injection, which was confirmed by determining the Ti biodistribution via ICP-MS (Figure S9). After receiving nano TiO2 injection intravenously for 1 h, the mice were injected with rUCNP, and the luminescence signal in specific wavelengths was collected by luminescence imaging. A strong luminescence signal was observed from the liver because of the high liver accumulation/retention of rUCNP (Figure 4F). Stronger green luminescence was found in the nano TiO2 free mice than that receiving nano TiO2 injection. To further ascertain that ratiometric ·OH detection could be identified by luminescence imaging in vivo, the luminescence signal intensity at 800 nm and 540 nm was calculated and analyzed, respectively, as well as the 800 nm/540 nm luminescence ratio by randomly selecting areas in the liver (Figure 4G). A high 800 nm/540 nm ratio (3.35:1) was obtained from the liver of mice receiving nano TiO2 injection, which was almost 2-fold higher than the nano TiO2 free ACS Paragon Plus Environment

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mice. These finding provided solid evidence that rUCNP could be used to detect ·OH in vitro and in vivo by ratiometric luminescence intensity analysis to evaluate nano TiO2-induced oxidative stress. Toxicity studies Although many works have illustrated the safety of rare-earth upconversion nanoprobes for bioapplications, a toxicity study of novel rUCNP both in vitro and in vivo is required. The 3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used for a cytotoxicity study. After incubation with various concentrations of rUCNP, CCC-HEL-1 cells showed high cellular viabilities (> 90%), suggesting that there was no significant viability difference between cells in the absence/presence of rUCNP (0–1.0 mg mL–1) within 24 and 48 h (Figure S10A). Furthermore, cellular reactive oxygen species determination by singlet oxygen sensor green showed no observable reactive oxygen species overproduction in cells incubated with rUCNP (Figure S10B). These results suggested that rUCNP has low cytotoxicity and does not induce detectable cellular oxidative stress. To get more data regarding the safety of rUCNP, toxicity was further studied in vivo. Healthy mice were given rUCNP (20 mg kg–1) by intravenous injection and monitored for 30 days. No death or abnormal behavior was observed in the mice receiving rUCNP injection. Moreover, complete blood panel tests and serum biochemistry assays were performed to study the influence of rUCNP on liver and kidney functions. Indices including alanine aminotransferase, aspartate aminotransferase, total bilirubin, total protein, albumin, and creatinine were tested. No significant differences between mice receiving PBS injection and rUCNP injection were found, suggesting that no notable liver or kidney function disturbances were induced by rUCNP (Figure S10C,D). Additionally, hematoxylin–eosin stained histological examinations of major organs, including heart, liver, spleen, lung, and kidney, demonstrated that rUCNP had no perceptible adverse effects within 30 days post-injection



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(Figure S10E). These systemic administrations provide solid evidence for the safety of rUCNP for bioapplications at our tested dose.

Conclusions We developed a novel upconversion nanoprobe via a facile complexation method for ·OH detection. As FeII in the 4-amino salicylic acid-based complex can be oxidized to FeIII by ·OH, our nanoprobe showed strong absorbance in the presence of ·OH, which resulted in the generation of energy transfer and the selective quenching of luminescence in the visible region. Utilizing the different response levels of luminescence centered at 540 nm and 800 nm to ·OH concentration, a ratiometric luminescence detection method was proposed for further application, with a lower detection limit of 2 nM and broad linear range from 4 nM–16 µM. More importantly, the ·OH generated from nano TiO2-induced oxidative stress was also successfully detected by ratiometric luminescence both in hepatocytes and in a mouse model. Higher 800 nm/540 nm luminescence ratio was observed in the livers of mice receiving nano TiO2 injection than those TiO2-free injection. Moreover, it was noteworthy that the nanoprobe could easily be recycled after ·OH detection by treatment with reducing agents. Our work provided a novel strategy for the development of recyclable upconversion nanoprobe via a facile preparation method for molecule detection and took a further step towards the application of molecule detection that was adapted for different biological environments.

Acknowledgements The authors thank the funding of National Natural Science Foundation of China (21301121),

Beijing

talent

foundation

outstanding

young

individual

project

(2015000026833ZK02), and Youth innovative research team of Capital Normal University.

Supporting Information ACS Paragon Plus Environment

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Supporting Information is available free of charge on the ACS Publications website, including detailed experiment section; EDXA and XRD of NaLuF4:Yb,Er, Tm nanoparticles; Zeta potential of NaLuF4:Yb,Er,Tm upconversion nanoparticles and rUCNP; TEM images, DLS, UV-vis-NIR spectra, and UCL spectra of rUCNP before and after 50 times recycle; Relationship between 800 nm/540 nm, 654 nm/540 nm, and 800 nm/654 nm with the ·OH ; Luminescence response of rUCNP solution in the aqueous containing various ROS; TEM image, XRD pattern, and size distribution of nano TiO2; DLS of nano TiO2 and nano TiO2@BSA; Biodistribution of nano TiO2 within 1 hour; MTT assay; serum biochemistry results; tissue sections; equation for the calculation of FRET efficiency.

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Zhang, F. Anal. Chem. 2017, 89, 3492-3500. (29) Chen, Z.; Liu, Z.; Li, Z.; Ju, E.; Gao, N.; Zhou, L.; Ren, J.; Qu, X. Biomaterials. 2015, 39, 15-22. (30) Li, Z.; Liang, T.; Lv, S.; Zhuang, Q.; Liu, Z. J. Am. Chem. Soc. 2015, 137, 11179-11185. (31) Mei, Q.; Li, Y.; Li, B. N.; Zhang, Y. Biosensor. Bioelectro. 2015, 64, 88-93. (32) Guo, Q.; Liu, Y.; Jia, Q.; Zhang, G.; Fan, H.; Liu, L.; Zhou, J. Anal. Chem. 2017, 89, 4986-4993. (33) Su, Q.; Feng, W.; Yang, D.; Li, F. Accounts Chem. Res. 2017, 50, 32-40. (34) Deng, R. R.; Xie, X. J.; Vendrell, M.; Chang, Y. T.; Liu, X. G. J. Am. Chem. Soc. 2011, 133, 20168-20172. (35) Peng, J.; Samanta, A.; Zeng, X.; Han, S.; Wang, L.; Su, D.; Loong, D. T. B.; Kang, N. Y.; Park,S. J.; All, A. H.; Jiang, W.; Yuan, L.; Liu, X. G.; Chang, Y. T. Angew. Chem. Int. Ed. 2017, 56, 4165-4169. (36) Ye, X. C.; Collins, J. E.; Kang, Y. J.; Chen, J.; Chen, D. T. N.; Yodh, A. G.; Murray, C. B. Proc. Natl. Acad. Sci. USA. 2010, 107, 22430-22435. (37) Liu, Y.; Guo, Q.; Zhu, X.; Feng, W.; Wang, L.; Ma, L.; Zhang, G.; Zhou, J.; Li, F. Adv. Funct. Mater. 2016, 26, 5120-5130. (38) Dong, A. G.; Ye, X. C.; Chen, J.; Kang, Y. J.; Gordon, T.; Kikkawa, J. M.; Murray, C. B. J. Am. Chem. Soc. 2010, 133, 998-1006. (39) Oushiki, D.; Kojima, H.; Terai, T.; Arita, M.; Hanaoka, K.; Urano, Y.; Nagano, T. J. Am. Chem. Soc. 2010, 132, 2795−2801. (40) Muhr, V.; Wurth, C.; Kraft, M.; Buchner, M.; Baeumner, A. J.; Resch-Genger, U.; Hirsch, T. Anal. Chem. 2017, 89, 4868-4874. (41) Li, X. L.; Peng, Q.; Yi, J. X.; Wang, X.; Li, Y. D. Chem–Euro. J. 2006, 12, 2383-2391. (42) Zheng, X. S.; Hu, P.; Cui, Y.; Zong, C.; Feng, J. M.; Wang, X.; Ren, B. Anal. Chem. 2014, 86, 12250-12257. ACS Paragon Plus Environment


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Scheme 1. The schematic of ratiometric ·OH detection based on recyclable nanoprobe by FRET between NaLuF4:Yb,Er,Tm and 4-ASA-Fe(III).


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Figure 1. The characteristic of rUCNP. A) TEM image and size distribution (insert) of NaLuF4:Yb,Er,Tm nanoparticles. B) HR-TEM image and the corresponding Fouriertransform diffraction pattern (insert) of NaLuF4:Yb,Er,Tm nanoparticles. C) FTIR spectra of NaLuF4:Yb,Er,Tm nanoparticles and rUCNP. D) UV-vis-NIR spectra and photograph (insert) of rUCNP reacted with different ·OH . E) UV-vis-NIR spectra and photograph (insert) of rUCNP, oUCNP, and oUCNP treated with GSH and AA. F) Monitoring 800 nm/540 nm of rUCNP recycled 50 times.



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Figure 2. The mechanism of ratiometric ·OH detection based on rUCNP. A) The proposed energy transfer processes responsible for the rUCNP. Vertical and wavy arrows represent non-radiative transitions, curved arrows represent non-radiative energy transfer, and colored arrows represent emissions. Clear overlap of absorbance wavelength and luminescence emission range can be observed, which will result in the FRET generation between the 4-ASA-Fe(III) and Er3+ (2H11/2→4I15/2, 4H3/2→4I15/2), as well as Tm3+ (1G4→3H6), though the blue emission was too weak to be observed on UCL spectra. B) UV-vis-NIR and UCL spectra of rUCNP.


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Figure 3. The ·OH detection capacity of rUCNP. A) Luminescence life time of rUCNP in the absence and presence of ·OH. B) UCL spectra of rUCNP reacted with different concentration of ·OH. C) The linear relationship between ·OH with the 800 nm/540 nm in the range from 4 nM to 16 µM. D) Corresponding Commission Internationale de l’Eclairage (CIE) chromaticity coordinates and photograph (insert) of luminescence from the samples shown in Figure 3B. E) Luminescence response of rUCNP solution in the aqueous containing different final concentration of ions, amino acid, and biomolecules: 1 mM for Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Ni2+, Zn2+, Cl-, and NO3-, 10 µM for Val, Gly, Cys, Glu, Lys, and 100 µM for Glc, GSH, DA, DOPAC, AA, and BSA. Luminescence response of rUCNP solution in the aqueous containing 1 µM ·OH was used for normalization.



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Figure 4. The ratiometric detection of ·OH generated from the oxidative stress induced by Nano TiO2. A) The diagram of in vitro ·OH detection and evaluation of Nano TiO2induced oxidative stress level. The ·OH within 12 h in live nano TiO2-incubated hypatocyte with or without light B), in live hypatocyte incubated with different nano TiO2 concentration C), and in live hypatocyte incubated with nano TiO2 and nano TiO2@BSA D). The first 5 h in each experiment was nano TiO2 free. Live hypatocyte without nano TiO2 incubation was used as blank group. E) The diagram of ·OH ratiometric imaging in vivo. F) Bright field, luminescence, color-mapped luminescence, and merge images of mice with and without receiving nano TiO2 injection. G) The box chart of luminescence intensity and 800 nm/540 nm ratio (insert) in liver region obtained from Figure 4F.


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Table 1. Comparison of the recyclable rUCNP and previous reported nanoprobes for hydroxyl radical detection. Acceptor

LOD (M)

4-ASA-Fe(II)

2×10

Rhodamine B

3×10

Carminic acid

2.1×10

Indocyanine green

4×10

-9

Linear range (M) -9

-5

4×10 -1.6×10

-8

0-1.25×10 -7

-12

-5

-5

0-1×10 1.6×10

-11

- 2×10

-6

Recyclable

Reference

yes

this work

no

29

no

31

no

32



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For ToC only.


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