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Dual-activator codoped upconversion nanoprobe with core-multishell structure for in vitro and in vivo detection of hydroxyl radical Xin-Yue Song, Jiayu Zhang, Zihong Yue, Zonghua Wang, Zhihong Liu, and Shusheng Zhang Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b02995 • Publication Date (Web): 18 Sep 2017 Downloaded from http://pubs.acs.org on September 19, 2017

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Analytical Chemistry

Dual-activator codoped upconversion nanoprobe with coremultishell structure for in vitro and in vivo detection of hydroxyl radical Xinyue Song,a Jiayu Zhang,a Zihong Yue,b Zonghua Wang,b Zhihong Liu*c and Shusheng Zhang*a Shandong Provincial Key Laboratory of Detection Technology for Tumour Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China. b Shandong Sino-Japanese Center for Collaborative Research of Carbon Nanomaterials, College of Chemistry and Chemical Engineering, Qingdao University, Shandong 266071, P. R. China. cKey laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, P. R. China. *Corresponding Author: Zhihong Liu, Fax: 86-27-68754067, E-mail: [email protected]; Shusheng Zhang, Fax: 86-5398766107, E-mail: [email protected]. a

ABSTRACT: Monitoring the fluctuation of hydroxyl radical (•OH) in the body can serve as an effective tool for the prediction of relative diseases, which, however, is highly challenging due to its short lifetime, high reactivity and extremely low concentration. Sandwich structured lanthanide-doped upconversion nanoparticles (UCNPs) exhibit unique luminescence properties and great prospects in bioimaging. Nonetheless, their rather low luminescence efficiency and intensity are serious limitations for their application. Herein, we report on dual-activator codoped UCNPs with a core-multishell structure that greatly improve the luminescence intensity and lifetime by 46-fold and 2.6-fold, respectively, than those of the mono-activator doped sandwich structured UCNPs. Moreover, emitting ions in the designed CMS-UCNPs were confined in a homogenous and thin shell layer (~2 nm), thus the luminescence resonance energy transfer (LRET)-based CMS-UCNPs@azo dye nanoprobe exhibited a largely shortened energy transfer distance and a pronounced luminescence quenching yield (97%), affording the nearly zero background signal and achieving an ultrahigh sensitivity for the detection of •OH (with LOQ of 0.10 fM). With good biocompatibility, low biotoxicity, enhanced luminescence intensity and lifetime, the developed nanoprobe was competent in monitoring the subtle fluctuation of •OH concentration both in vitro and in vivo.

Hydroxyl radical (•OH) is the most potent oxidant and aggressive free radical among the reactive oxygen species (ROS) and could attack various biological species at extremely high rates, leading to cell disorders, damage and even death.1 Its abnormal levels in living cells often result in numerous diseases such as inflammation, neurodegenerative disorders and cardiovascular disease, even cancer.2 Therefore, it is urgently demanded to exploit highly selective and sensitive methods for real-time and in vivo monitoring of •OH. Compared with other determination methods, fluorescence probes own several outstanding advantages such as high specificity and sensitivity, localized information at the target site and real-time monitoring.3,4 Till now, various fluorescence probes have been developed for the in vitro and in vivo determination and bioimaging of ROS.5-7 Superior to conventional single-photon excited fluorescent materials such as organic fluorophores,8,9 metal complexes,10,11 and semiconductor quantum dots,12,13 lanthanide-doped upconversion nanoparticles (UCNPs) are able to convert continuous near-infrared (NIR) excitation wave to ultraviolet, visible, or NIR emissions via a two-photon or multi-photon mechanism.14 Therefore, they exhibit unique luminescent properties such as narrow and tunable emission bands, large anti-Stokes shift, long lifetime, high photostability, low toxicity, no photobleaching and no blinking.15 Moreover, the

excitation light of UCNPs is coincident with the biological transparency window, allowing greater tissue penetration depth and reduced background autofluorescence.16 With these outstanding merits, they have widely been employed in in vitro microscopy,17 multi-model imaging in small animals and drug carrier.18 Based on luminescence resonance energy transfer theory (LRET), UCNPs have widely been used to develop nanoprobes since they could act as the energy donor and their luminescence could be quenched by specific target recognizing moieties as the energy acceptor.19 It is widely known that the energy-transfer efficiency is mainly depended on two factors, i.e., the donor-to-acceptor distance and the spectral match (the emission of energy donor and the absorption of energy acceptor).19 The spectral match could be achieved by choosing suitable lanthanide ions and recognizing moieties. Thus, the donor-to-acceptor distance becomes a critical factor for the enhancement of the energy transfer efficiency. Recently, Li et al. group developed a novel sandwich structured UCNPs (SWUCNPs) with a bared surface which consisted of an inert core and a thin layer with confined emitters close to an inert outer shell.19,20 Due to the largely shortened distance between the UCNPs and the energy acceptor, an improved luminescence quenching degree of up to >80% was obtained.

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However, since the luminescence layer was confined in a thin layer (around 2-3 nm), SWUCNPs exhibited relatively weeker luminescence efficiency and intensity compared with those with emitters dispersed in the whole nanoparticle. Therefore, the luminescence intensity of SWUCNPs should be further improved, which is critical and necessary for the application of UCNPs, especially in the deep-tissue imaging.21 Moreover, •OH has a nanosecond lifetime, high reactivity and extremely low in vivo concentration, which makes its determination quite challenging. Thus, it is also desired to further improve the luminescence quenching degree of UCNPs so as to obtain higher sensitivities. Trivalent rare-earth ions such as Y3+, Yb3+, Er3+, Tm3+ and Ho3+ are suitable candidates for the upconversion luminescence (UCL) process due to their abundant energy level and narrow emission spectral lines.14 So far, NaYF4 is considered as the most efficient host material owing to the high refractive index and low photon energy while Yb3+ acts as an effective sensitizer attributed to its large absorption cross section and long lifetime of 2F5/2 excitation level.14 Trivalent rare-earth ions Er3+ and Tm3+ have ladder-like arrangement energy levels and are frequently used as activators to generate the UCL emission under NIR excitation.14 Their matching energy levels enable several resonant and non-resonant energy transfers occur.22,23 Therefore, Tm3+ could act as an effective sensitizer, enriching the population of 2H11/2 and 4S3/2 level of Er3+.24,25 Additionally, Tm3+ ion could temporarily store the energy, inducing a three-photon process via excited state absorption and cross relaxations.26 Therefore, codoping of dual activators would act as an effective strategy to enhance the UCL intensity of UCNPs. Based on the above strategy, we prepared the coremultishell structured UCNPs (CMS-UCNPs), that was NaYF4@NaYF4:Yb,Tm@NaYF4:Yb,Er@NaYF4, by the successive layer-by-layer doping method. To construct a LRET based nanoprobe, the three shells were controlled with a thickness of around 2 nm to shorten the energy-transfer distance between UCNPs (acting as the energy donor) and a specific azo dye for selective recognition of •OH (acting as the energy acceptor). Such structure would guarantee a high LRET efficiency because this process generally occurred within a distance of less than 10 nm. The newly developed CMS-UCNPs@azo dye nanoprobe was applied to detect •OH concentration both in vitro and in vivo. Moreover, its distribution and real-time monitoring of •OH in cancer cells, biotoxicity, biodistribution, in vivo detection of •OH in LPSinduced acute inflamed mice and tumour-bearing mice were investigated. EXPERIMENTAL SECTION Synthesis of Oleic Acid Protected CMS-UCNPs. In general, dopants concentration should be optimized to get the highest efficiency of UCL. The higher or lower doping concentrations were proved to be non-beneficial to the promotion of luminescence efficiency, since higher doping concentration could induce the cross-relaxations between the dopants while the lower doping concentration could cause the reduction of the luminescent centre.27,28 Thus, in this experiment, the established optimum doping ratio for the Y3+, Yb3+ and Er3+ codoped system (80:18:2 in molar) was used to prepare CMS-UCNPs. Oleic acid (OA) stabilized core-multishell structured NaYF4@NaYF4:Yb,Tm@NaYF4:Yb,Er@NaYF4 was

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synthesized using a layer-by-layer seed-mediated shell growth strategy.19,20,29 The detailed procedures were as follow: Firstly, Y(oleate)3 and Ln(oleate)3 were prepared according to a previously reported method,20 respectively, and then dissolved in OA/1-octadecene (ODE) mixing solution (V:V=1:1) via stirring to obtain the final concentration of 0.2 mmol/mL. Secondly, 1 mmol Y(oleate)3 and 20 mmol NaF were dispersed in 20 mL of OA/ODE mixing solvent (V:V=1:1). Then, the reaction system was degassed to remove residual water and oxygen followed by aerating with argon (Ar) to avoid the oxidation of OA ligand. NaYF4 core nucleated at 110oC for 60 min and further grew at 340oC for 90 min. After 4 mL of the above reaction mixture was retrieved for characterization, 0.4 mmol Ln(oleate)3 (Y:Yb:Tm=80-x:20:x in molar) in 8 mL of OA/ODE (V:V=1:1) mixture was injected into the reaction and reacted at 340oC for 20 min to form the first luminescent shell on the surface of NaYF4 core. After another 6 mL of the reaction mixture was taken out for characterization, 0.4 mmol Ln(oleate)3 (Y:Yb:Er=80:18:2) in 8 mL of OA/ODE (V:V=1:1) mixture was added to grow the second luminescent shell. After 20 min, 8.5 mL of the reaction mixture was taken out and then 0.4 mmol Y(oleate)3 in 8 mL of OA/ODE mixing solvent (V:V=1:1) was injected to prepare the outer shell NaYF4. 20 min later, the reaction mixture was cooled to the room temperature. The prepared CMS-UCNPs was precipitated by two-fold volume of ethanol, centrifugally collected and washed with hexane/ethanol (V:V=1:6) for several times. The finally collected CMS-UCNPs was dispersed in hexane and stored at -20oC for further use. Preparation of Bared CMS-UCNPs. The exterior OA ligands could interfere with the coordination interaction between the CMS-UCNPs and recognition moieties of targets, as well as increase the LRET distance, so it was removed through the acid treatment.19,20,30 Briefly, 60 mg of the OA protected CMS-UCNPs was centrifuged from hexane and then dispersed in 30 mL of acidic ethanol solution (pH=1) under ultrasonification for 1 h. Bared nanoparticles were collected by centrifugation and washed with an acidic ethanol solution (pH=4), ethanol and ultrapure water for several times and finally redispersed in ultrapure water for storage. Construction of CMS-UCNPs@Azo dye Nanoprobe. 0.30 mg of bared CMS-UCNPs was mixed with 5.0-180 nmol azo dye in 3-(N-morpholino)propanesulfoinc acid (MOPS) buffer (1.0 mL, 10 mM, 100 mM KCl, pH=7.2) and then the mixture was shaken gently for overnight. The azo dye covered CMS-UCNPs was centrifuged and washed with the above MOPS buffer for several times until there was no free azo dye in the washing solution. Finally, CMS-UCNPs@azo dye nanoprobe was redispersed in MOPS buffer with a concentration of 0.30 mg/mL. RESULTS AND DISCUSSION Properties of the Core-Multishell Structured UCNPs. In this work, we designed a core-multishell structured UCNPs for the first time which was composed of NaYF4@NaYF4:Yb,Tm@NaYF4:Yb,Er@NaYF4. In a typical procedure, NaYF4 nanocrystals were first synthesized as the core, then coated with NaYF4:Yb,Tm and NaYF4:Yb,Er as the first shell and the second shell, respectively. Finally, outer shell NaYF4 was grown on the surface of the luminescent layers to prevent from environmental quenching. The transmission electron microscopy (TEM) images of the assynthesized nanoparticles (Figure S1a-d) illustrated the


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uniform size of UCNPs and their evolution (from the ∼22.02 ± 0.8 nm NaYF4 cores to the ∼27.26 ± 1.2 nm Tm doped core-shell UCNPs, further to the ∼31.16 ± 1.6 nm Tm3+ and Er3+ codoped core-double shell UCNPs, and ultimately to the 35.1 ± 1.3 nm NaYF4 protected core-multishell UCNPs). Guided by the layer-by-layer seed-mediated shell growth strategy, the thickness of each shell could be controlled as thin as 2 nm. The crystalline structure and phase purity of the obtained nanoparticles were determined by X-ray powder diffraction (XRD). As shown in Figure S2, the XRD pattern of the asprepared core and core-shell structured UCNPs can be indexed to pure hexagonal phases (JCPDS no. 16-0334). As shown in Figure S3 and Figure S4, the prepared CMSUCNPs with different doping concentration of Tm3+ displayed similar particle size and hexagonal phase. In terms of their UCL intensity, the introduction of a Tm3+ doped luminescent layer largely increased the UCL intensity (Figure 1a). Moreover, with the doped concentration of Tm3+ increased to 0.8%, the UCL intensity of the CMS-UCNPs was increased by 46-fold, which proved the effective energy transfer between Tm3+ and Er3+ (Figure 1b).22,23 Additionally, the codoping of Tm3+ and Er3+ increased the lifetime of the prepared CMSUCNP from 149.7 μs (0%Tm) to 392.8 μs (0.8%Tm) (Figure S5). Compared with the developed SWUCNPs,19,20 the novelly designed CMS-UCNPs with dual activators not only favored the LRET via the shorted energy transfer distance but also displayed obviously enhanced UCL intensity and longer lifetime, which was quite meaningful for the application of UCNPs, especially in the small animal imaging. Thus, such homogenous layer-by-layer seed-mediated shell growth and dual-activator codoping strategy are effective ways to obtain high-quality UCNPs with advanced nanostructure and optical properties by engineering the local distribution of the host, sensitizer and activator in a microcosmic.31 Design Principle of CMS-UCNPs@Azo dye Nanoprobe for the •OH Determination. The developed upconversion nanoprobe for the determination of in vitro cytosol •OH in HeLa cell and in vivo •OH in inflamed mice and tumourbearing mice was described in Scheme 1.

Scheme 1: Schematic illustration of the designed principle of the CMS-UCNPs@azo dye nanoprobe for in vitro and in vivo determination of •OH.

In terms of the recognition ligand for the determination of •OH, an azo dye (2-hydroxy-1-(2-hydroxy-4-sulfo-1naphthylazo)-3-naphthoic acid) was chosen in consideration of its specific reaction with •OH (oxidation and decomposition by •OH), good spectral match with the prepared UCNPs at 545 nm, stable assembly on the bared surface of the prepared UCNPs (through the chelation interaction), as well as negligible pH interference (Figure S6). The chemical structure of the azo dye and its molar extinction coefficient at 545 nm (7602 L mol-1 cm-1) were shown in Figure S7. To construct the LRET-based fluorescence probe, the above prepared CMS-UCNPs were used as the energy donor while the chosen azo dye acted as both the energy acceptor and recognition site for •OH. After selectively reacting with •OH, the absorbance value of the azo dye at 545 nm was correspondingly decreased, leading to decreased quenching efficiencies and turn-on fluorescence signals which were quantitatively related to •OH concentration.



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Figure 1: (a) The normalized UCL intensity of CMS-UCNPs with different doped concentration of Tm3+; (b) The relative UCL intensity of CMS-UCNPs at λ=545 nm with different doped concentration of Tm3+; (c) The absorbance of azo dye and oxidized product; the fluorescence of UCNPs and azo dye modified UCNPs; (d) Relative fluorescence intensity F/Fo and photos of the nanoprobe with different concentrations of the azo dye; F and Fo represented the UCL emission intensity in the presence and the absence of the azo dye, respectively.

Fabrication of the CMS-UCNPs@Azo dye Nanoprobe. To further favour the LRET process, acid treatment was used to remove the oleate ligands and obtain a bared surface. As shown in FTIR analysis (Figure S8), the characteristic IR peaks of oleic acid (2925 and 2857 cm-1 attributed to the intense asymmetric and symmetric stretching vibrations of CH bond of CH2 group) disappeared after the acid treatment, which proved the successful removal of the oleate ligands. As shown in Figure S9, the acid treatment did not affect the shape, size and the crystalline state of the UCNPs, moreover, its UCL did not display significant variations in 2 h. After the removal of the oleate ligands, the azo dye was tagged onto the bared surface of CMS-UCNPs by chelate interaction. The successful fabrication of CMS-UCNPs@azo dye nanoprobe was confirmed by the appearance of the characteristic IR peaks of the azo dye and the change of ζ potential (Figure S10). Owing to the well-matched spectra and effective energy transfer distance, an effective LRET and high quenching yield as high as 97% were expected (Figure 1c-d) which were quite impressive and prior to those obtained by previously reported SWUCNPs nanoprobes.19,20 The Response of the CMS-UCNPs@Azo dye Nanoprobe to •OH in Aqueous Solution. NaNO3 aqueous solution was photolyzed to produce •OH of femtomolar (fM) concentrations under the illumination of an UV lamp.32-34 Different concentrations of •OH were obtained by illuminating the corresponding amount of NaNO3 for constant time. Based on the reaction rate equation and the fluorescence properties of sodium salicylate, •OH with the concentration range of 0.10163.20 fM was obtained by the photolysis of 0.005-250 mM NaNO3 (Figure S11). The azo bond of the azo dye was decomposed by •OH and its absorbance at 545 nm was obviously decreased (Figure S12). Therefore, the energy transfer from the UCNPs to the azo dye was weakened and the luminescence of UCNPs was corresponding recovered, producing sensitive turn-on signals for the determination of • OH. (Figure 2a). To investigate the response of the CMS-

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UCNPs@azo dye nanoprobe to •OH, the prepared nanoprobe was added into various concentrations of NaNO3 (0.005-250 mM) solution with the final concentration of 0.24 mg/mL. And then, the above mixture was irradiated under an UV lamp for 30 min. The UCL intensities at 545 nm were recorded after excited with continuous wave laser at 980 nm. A linear correlation equation y=0.137x (correlation coefficient R2=0.9994) was obtained between the relative UCL intensity of the probe (F-Fo)/Fo and the concentration of •OH (Figure 2b). The quantification limit (0.10 fM) was lower by one order of magnitude than that obtained by the ever developed mOGSWUCNPs nanoprobe19 which was quite meaningful for the determination of biological •OH in vivo. To study the selectivity of the prepared nanoprobe, various potential interfering biological species, such as ROS (1O2, O2 •-, H2O2, OCl-), metal ions (K+, Mg2+, Ca2+, Fe3+, Cu2+, Zn2+), amino acids (cysteine, glycine, histidine), BSA (bovine serum albumin), glucose, glutathione and lysozome were examined in parallel under the same experimental conditions. As shown in Figure 2c, a remarkable enhancement of (F-Fo)/Fo was induced by •OH while no obvious changes were observed for other physiologically interfering species including other ROS, metal ions and some biological species, demonstrating the high specificity of the probe.

Figure 2: (a) UCL spectra of the prepared CMS-UCNPs@azo dye nanoprobe with different concentrations of •OH; (b) Relative UCL intensity value (F-Fo)/Fo with different concentrations of •OH, F and Fo represented for UCL intensity in the presence and absence of •OH; (c) Relative UCL intensity value (FanalyteFo)/(Fhydroxyl radical-Fo) with different interfering species.

Monitoring •OH in Living Cells. Then, the biological application of the CMS-UCNPs@azo dye nanoprobe was investigated by monitoring intracellular •OH in living cells. First, its weak cytotoxicity was validated by the MTT assay which showed larger than 91.5% of the cell viability at the concentration of 0.5 mg/mL (Figure S13). Then, the biological application of the CMS-UCNPs@azo dye nanoprobe was investigated by monitoring intracellular •OH in living cells. First, its distribution was studied under different incubation times (Figure S14). The nanoprobe was first mainly assembled on the cell membrane and in the interstitial substance, and then uptaken into the cytosol via an endocytosis process, along with the incubation,


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demonstrating the good biocompatibility of the prepared nanoprobe. As shown in Figure 3a, the nanoprobe could serve as an effective tool to detect intracellular •OH in living cells. Intracellular concentration of •OH could be increased by the treatment of phorbol myristate acetate (PMA) reagent which simulates HeLa cells to produce ROS by respiratory burst.35 And the intracellular concentration of •OH would be decreased by the treatment of 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), a widely-used radical scavenger, thus PMA and TEMPO reagents were used to change the concentration levels of intracellular •OH in living cells. As shown in Figure 3b, an obvious increase in the UCL intensity was observed after HeLa cells were treated with PMA. As expected, the UCL intensity was obviously decreased when HeLa cells were stimulated with TEMPO reagent before treated with PMA (Figure 3c). Therefore, the developed CMS-UCNPs@azo dye could achieve the real-time monitoring of •OH in cancer cells.

Figure 3: Confocal microscopic images of HeLa cells (a) incubated with the 0.24 mg/mL of CMS-UCNPs@azo dye nanoprobe; (b) pretreated with 500 ng/mL of PMA before incubated with the CMS-UCNPs@azo dye nanoprobe; (c) pretreated with 1.0 mM of TEMPO, then treated with PMA and the CMS-UCNPs@azo dye nanoprobe; (d) Normalized average UCL intensities in (a)-(c). Images were collected at 510-570 nm.

Monitoring •OH in Small Animals. To check the applicability of the developed CMS-UCNPs@azo dye nanoprobe for in vivo detection of •OH, we designed two animal models, i.e., LPS-induced acute inflamed mice and tumour-bearing mice, which were both reported to have elevated level of •OH in organs. As shown in Figure S15, no toxicity sign was observed from the animals injected with 4.0 mg/100 g CMS-UCNPs@azo dye nanoprobe as compared to those treated with physiological saline. The investigation results of the bio-distribution of the nanoprobe in mice showed that the developed nanoprobe was mainly accumulated in kidney, intestine and liver, whereas little accumulation was found in the heart and lung (Figure S16). To further study the kinetic characteristic of the prepared nanoprobe in living animals, 4.0 mg/100 g nanoprobe was intraperitoneally (i.p.) injected into mice which was treated with 3.0 mg/100 g LPS for 24 h. As shown in Figure 4a, the UCL intensity of the prepared CMS-UCNPs@azo dye nanoprobe reached its maximum as the incubation time was prolonged to 12 h. For the in vivo determination of •OH in small animals, we set four batches of test samples. First, a blank control was designed where the inflamed mice was injected with only physiological saline. This sample showed near-zero signal under the excitement of 980 nm laser (Figure 4b), demonstrating the negligible autofluorescence of the

biological sample. For other samples, 4.0 mg/100 g nanoprobe was injected into the mice with different inflammation symptoms induced by 1.0 mg, 2.0 mg and 3.0 mg/100 g LPS, respectively. As shown in Figure 4b, the UCL intensity increased along with the amount of LPS used, demonstrating the capability of the prepared nanoprobe to perform in vivo •OH detection.

Figure 4: (a) Confocal microscopic images of the liver slices of LPS mode mice after incubated with the prepared nanoprobe for different incubation time; (b) Confocal microscopic images of the liver slices of LPS mode mice injected with 0.0 mg/100 g, 1.0 mg/100 g, 2.0 mg/100 g and 3.0 mg/100 g of LPS 24 h before the injection of 4.0 mg/100 g of the prepared nanoprobe; (c)-(d) Normalized average UCL intensities in (a)-(b). Images were collected at 510-570 nm.

To explore whether the CMS-UCNPs@azo dye nanoprobe could be activated within the target tumour and achieve specific tumour section imaging with high signal-tobackground ratio, fluorescence imaging was performed in the tumour-bearing mice. After circulation and metabolism for 12 h in the living body, the CMS-UCNPs@azo dye nanoprobe was delivered to tumour sites via enhanced permeation and retention, and clear fluorescence was observed due to the existence of elevated concentration of • OH (Figure 5). Little fluorescence was observed in other organs including intestines, lung, kidney and heart, owing to low accumulation or quite low concentration of •OH in these normal tissues (Figure S17). Thus, there was a very high signal-to-background ratio between tumour and normal tissues. The results proved that the prepared CMS-UCNPs could gather in tumour, proving its ability to target tumours.



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Shusheng Zhang*: Shandong Provincial Key Laboratory of Detection Technology for Tumour Markers, College of Chemistry and Chemical Engineering, Linyi University, Linyi 276005, P. R. China, Tel: 86-539-8766107; Fax: 86-539-8766107; E-mail: [email protected] (S.S. Zhang).

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

Figure 5: Confocal microscopic images and the relative UCL intensity of the tumour slices of the tumour-bearing mice after incubated with the prepared nanoprobe for 12 h. Images were collected at 510-570 nm; The information of the above tumourbearing mice was shown in the table.

CONCLUSION In summary, an ultrasensitive upconversion nanoprobe has been developed for the monitoring of •OH fluctuation in cancer cells and small animals. Guided by the layer-by-layer seed-mediated shell growth strategy, an inert NaYF4 core was coated with two activators doped luminescent shells and then coated by a NaYF4 layer which protected the luminescence from environmental quenching. The dual activator codoped core-multishell structured upconversion nanoparticles (UCNPs) increased the upconversion luminescence intensity and lifetime by 46-fold and 2.6-fold, respectively, than that of the mono-activator doped UCNPs. Taking advantage of the controlled thin shell layers and bared surface, the asconstructed UCNPs@azo dye nanoprobe showed a pronounced efficiency of luminescence resonance energy transfer, resulting in near-zero background and high quenching yield (as high as 97%). With ultralow quantification limit (0.10 fM) and good selectivity, the nanoprobe achieved the detection of •OH in living cancer cells and small animals with inflammation and tumour.

ASSOCIATED CONTENT Supporting Information Supporting information includes (1) chemicals and materials, (2) instrumentation, (3) TEM images of the CMS-UCNPs, (4) XRD images of the CMS-UCNPs, (5) TEM images of the CMS-UCNPs with different doping concentration of Tm3+, (6) XRD images of the CMS-UCNPs with different doping concentration of Tm3+, (7) lifetime measurement, (8) Effects of pH, (9) chemical structure and absorbance value of the azo dye, (10) characterization results of CMS-UCNPs@azo dye nanoprobe, (11) calculation of • OH, (12) absorbance change of the azo dye reacting with • OH, (13) MTT assay, (14) detection of cytosol • OH in HeLa cells and fluorescence imaging, (15) animal experiments.

AUTHOR INFORMATION Corresponding Author Zhihong Liu*: Key laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Science, Wuhan University, Wuhan 430072, P. R. China, Tel: 0086-13797010269; [email protected] (Z.H. Liu);

This work has been financially supported by the National Natural Science Foundation of China (Nos.: 21605068, 21535002, 21775063), the Key Project of Research and Development Plan of Linyi City (No. 2016GG027), the Natural Science Foundation of Shandong Province (No. ZR2016QZ001) and Primary Research and Developement Plan of Shandong Province (2017GGX40110).

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