Interfere-free Detection of Hydroxyl Radical and Arthritis Diagnosis by

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Interfere-free Detection of Hydroxyl Radical and Arthritis Diagnosis by Rare Earth-based Nanoprobe Utilizing SWIR Emission as Reference Qi Jia, Yuxin Liu, Yuai Duan, and Jing Zhou Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02855 • Publication Date (Web): 02 Aug 2019 Downloaded from pubs.acs.org on August 2, 2019

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Analytical Chemistry

Interfere-free Detection of Hydroxyl Radical and Arthritis Diagnosis by Rare Earth-based Nanoprobe Utilizing SWIR Emission as Reference

Qi Jia, Yuxin Liu, Yuai Duan, and Jing Zhou* Department of Chemistry, Capital Normal University, Beijing 100048, People’s Republic of China E-mail: Jingzhou@cnu. edu. cn

ABSTRACT: Due to the high oxidative potential of the hydroxyl radical (·OH), the accumulation of ·OH in tissues can cause inflammation, such as that in arthritis. Therefore, the development of ·OH detection with high efficiency and sensitivity is important for the treatment of related diseases. In this work, a cypate-modified core-shell NaErF4@NaLuF4 nanoprobe (csEr-Cy) was designed for detecting ·OH on the basis of a typical reaction between cypate and ·OH. The process resulted in the recovery of 654 nm upconversion luminescence emission of csEr because of a weakened inner filter effect (IFE) and Förster resonance energy transfer (FRET). The short-wavelength infrared (SWIR) emission at 1550 nm was not affected by ·OH addition; thus, enabling interference-free detection. Density functional theory (DFT) calculations were performed to explain the underlying mechanism. With the SWIR signal used as a reference for ·OH detection, the csEr-Cy nanoprobe showed higher sensitivity and penetration than visible reference. This method was successfully used 1 ACS Paragon Plus Environment

Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

in mice for the diagnosis of arthritis in vivo. Our results provide novel insights into improving the sensitivity of nanoprobes for molecule detection and disease diagnosis. Keywords: SWIR, hydroxyl radical, luminescence detection, cypate, arthritis

1. Introduction Aerobic cells produce a series of reactive oxygen species (ROS) during metabolic processes, and the accumulation of ROS causes various diseases, such as carcinogenesis and cardiovascular disease, as well as an inflammatory response.1-3 Among ROS, the hydroxyl radical (·OH) is the most harmful because of its high oxidative reactivity.4 Recent advances have indicated that ·OH plays an important role in inflammation, such as that in arthritis, which may causes physiological disorder in biological system. Arthritis is a kind of inflammatory disease that results in symptoms such as joint pain, swelling, stiffness, and a decreased range of motion of the affected joints.5,6 However, there are very little works concentrated on the diagnosis of arthritis. Therefore, the development of nanoprobes for highly sensitive and highly selective detection of ·OH is essential for the diagnosis of ·OH related disease, such as arthritis.7 Optical probes can be used for disease diagnosis and are suitable for the detection of ·OH in vivo. The primary types of luminescence probes include single-walled carbon nanotubes,8 small molecule dyes,9 and quantum dots.10,11 Compared with other probes, rare earth-based nanoprobes have been studied because of their advantages of 2 ACS Paragon Plus Environment

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the absence of photobleaching, adjustable multiple emission bands, absence of autofluorescence from biosamples, and high depth of tissue penetration.12-15 For example, Er3+ and Tm3+ indicating the transition of 4S3/2→4I15/2, 4F9/2→4I15/2, and 3H

3H

4→

6,

corresponding to 540 nm, 654 nm, and 800 nm emissions, respectively,

have been widely used in previous studies.16-18 By using proper ·OH responsive ligands, different types of rare earth-based nanoprobes can be designed for ·OH detection. In our previous studies, an ICG-modified NaLuF4:Yb,Er nanoprobe and a recyclable 4-ASA-Fe (II)-modified NaLuF4:Yb,Er, Tm nanoprobe were developed for ratiometric ·OH detection with good sensitivity and selectivity.19,20 By using one luminescence signal as a reference, highly sensitive ·OH detection can be achieved through ratiometric detection.21 However, traditional references are always in the visible (400–800 nm) and near-infrared (NIR; 800–1000 nm) region,6 and are easy influenced by ligands through Förster resonance energy transfer (FRET) and the inner filter effect (IFE);22 thus, decreasing the luminescence ratio with ·OH concentration and further decreasing probe sensitivity. Therefore, finding a signal that is not easily influenced for use as a reference should aid in improving ·OH detection. Recent studies have highlighted that, compared with visible or NIR luminescence, short-wavelength infrared (SWIR) (1300–1700 nm) has a greater penetration depth, spatial resolution, and a greater ability to decrease auto-luminescence signals and light scattering.23-31 Moreover, typical ·OH responsive ligands have no absorbance in the SWIR region and therefore, scarcely influence the SWIR intensity through FRET or IFE. Consequently, SWIR luminescence has the potential to serve as a better 3 ACS Paragon Plus Environment


reference.32 Er-doped luminescence nanoprobes with strong luminescence at 1550 nm though the 4I13/2→4I15/2 transition have been widely used as SWIR probes.33,34 Moreover, previous studies have indicated that cyanine-linked dyes, such as cypate substantially react with ·OH,19 thus, changing their characteristics of absorption in the range of 500 - 900 nm, but absorption in the SWIR region is not influenced. In this work, a cypate-modified core-shell NaErF4@NaLuF4 nanoprobe (csEr-Cy) was designed for detection of ·OH by using the SWIR signal at 1550 nm as a reference. On the basis of the reaction between cypate and ·OH, the emission of Er3+ at 654 nm was influenced by IFE and FRET, and the SWIR emission at 1550 nm was used as the reference for ratiometric detection, which was interference-free and more sensitive than traditional reference. On the basis of this detection, sensitive and selective ·OH detection was studied, and the csEr-Cy was investigated in mice for the diagnosis of arthritis.

2. Experimental section 2.1. Preparation of cypate modified core-shell NaErF4@NaLuF4 (csEr-Cy) The NaErF4 and the core-shell NaErF4@NaLuF4 (csEr) nanoparticles were prepared via a typical solvothermal method. After removed the oleic acid ligands on the surface by nitrosonium tetrafluoroborate, the bare csEr were obtained. The csEr was then modified by cypate in ethanol to obtain the final nanoprobe csEr-Cy and dispersed in deionization water. 4 ACS Paragon Plus Environment

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2.2. ·OH detection capacity study A typical Fenton reaction was used to generate hydroxyl radical (·OH). In the ·OH detection experiment, 1 mL of csEr-Cy, 0.5 mL of DMSO, 0.5 mL solution of various concentrations of H2O2 (0-500 μM), and 0.02 mL saturated solution of FeSO4 were mixed at room temperature and incubated for 5 min. The UCL and the SWIR spectra of the above samples were measured. To study the selectivity of csEr-Cy, the solution of H2O2 was changed into the solution of various ions (including Na+, K+, Ca2+, Mn2+, Fe2+, Zn2+, Cl-, CO32-, SO42-, NO3-, and PO43-) and reactive oxygen species (ROS), such as H2O2 and ClO-. The UV-vis-NIR spectra of the above samples were measured.

2.3. ·OH imaging and arthritis diagnose All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Beijing Vital River Laboratory Animal Technology Co., Ltd. and approved by the Animal Ethics Committee of the Vital River Institutional Animal Care and Use Committee (VR IACUC). Healthy BALB/c nude mice were used as a small-animal model for in vivo experimentation. The arthritis model on mice was established according to the previous work. Please see detail experimental section in Supporting Information.

3. Results and discussion 3.1. Synthesis and characteristics of csEr-Cy

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The core-shell, NaErF4@NaLuF4 (csEr), was synthesized via a typical solvothermal method. Transmission electron microscopy (TEM) indicated that the NaErF4 core had a narrow size distribution of 12.3 ± 2.9 nm and a uniform morphology (Fig. 1A and Fig. S1). After coating with NaLuF4, the diameter increased to 25.8 ± 4.5 nm, thus, suggesting the successful synthesis of csEr (Fig. 1B and Fig. S2). X-ray diffraction (XRD) patterns suggested that both the NaErF4 core and csEr had a highly crystalline hexagonal phase corresponding to the standard card of β-NaErF4 (JCPDS: 27-0689) and β-NaLuF4 (JCPDS: 27-0726) (Fig. 1D), respectively. High-resolution TEM (HR-TEM) also confirmed this finding (Fig. 1C). Electron diffraction X-ray analysis (EDXA) patterns illustrated the chemical composition of csEr (Fig. 1E). The csEr was then modified by cypate.35 Fourier transform infrared (FTIR) spectrum of csEr-Cy indicated bonds at 3232 cm-1 for R2-C=CH-R and 1637 cm-1 for C=C in phenyl, which were the same as the bonds of free cypate (Fig. S3, S4), thus, indicating that cypate, an ·OH responsive probe, was successfully modified on the surface of csEr. The csEr-Cy showed good solubility and stability in water (Fig. S5, S6), and a strong absorbance (500-900 nm, the absorption peak at 780 nm) was observed in the ultraviolet-visible-near infrared (UV-vis-NIR) spectrum of csEr-Cy (Fig. 1F). After the addition of ·OH, the absorbance peak decreased, thus, demonstrating that csEr-Cy could be used as an·OH responsive probe.

3.2. Ratiometric detection of ·OH by csEr-Cy To further study the reaction between csEr-Cy and ·OH, upconversion luminescence (UCL) and the short-wavelength infrared (SWIR) spectra were 6 ACS Paragon Plus Environment

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measured (Fig. 2A, B). The emissions at 527 nm, 540 nm, and 654 nm were observed in the UCL spectra under 980 nm excitation. As the concentration of ·OH (from 0 to 500 μM) increased, the UCL intensities increased, and the emission at 654 nm showed the most prominent increase. Moreover, after the addition of ·OH, the weak green luminescence changed to a strong red luminescence (Fig. 2A inset). The results suggested that csEr-Cy can be used as a nanoprobe for the detection of ·OH and that the emission at 654 nm can be used as the working signal. Moreover, according to the SWIR spectra, changes in the SWIR intensity of csEr-Cy at 1550 nm were scarcely observed, regardless of ·OH concentration (Fig. 2B). Therefore the ratiometric detection can be achieved by using 654 nm as working signal and 1550 nm as reference. To further determine whether 1550 nm might have better potential as an interference-free reference than 540 nm, a traditionally used reference wavelength, we compared the intensity changes at 540 nm and 1550 nm at the highest ·OH concentration of 500 μM. The intensity at 540 nm increased 731.92%, whereas that at 1550 nm increased only 1.93% (Fig. S7). Therefore, SWIR has the potential to be used as an interference-free reference for ·OH detection. The rate of the reaction and the selectivity of the detection were also investigated. The absorbance at 780 nm was determined and showed a clear decrease before reaching a plateau within 20 min (Fig. 2C); thus, suggesting a rapid reaction between csEr-Cy and ·OH. To investigate the selectivity of csEr-Cy, we added several ions (including Na+, K+, Ca2+, Mn2+, Fe2+, Zn2+, Cl-, CO32-, SO42-, NO3-, and PO43-) and ROS, such as H2O2 and ClO- into csEr-Cy, and determined the UV-vis-NIR spectra of 7 ACS Paragon Plus Environment


each sample. The absorbance of these ions and ROS remained as high as that of water and were significantly different from that of ·OH (Fig. 2D). It is also noteworthy that the analytical performance of csEr-Cy is comparable with previous reported nanoprobes (Table S1).36,37 These results suggested that csEr-Cy showed no obvious response to these ions and ROS, and hence had high selectivity in the detection of ·OH. csEr-Cy can react with ·OH rapidly and specifically, while using 654 nm as working signal and 1550 nm as an interfere-free reference may have the potential to apply in ratiometric imaging.

3.3. Improvement of sensitivity and penetration at 1550 nm To study whether the SWIR intensity at 1550 nm might be used to improve the sensitivity of ·OH detection, we used a traditional ratiometric detection method of ·OH with UCL at 540 nm as a reference for control. As the SWIR intensity at 1550 nm is merely changed, the intensity ratios of 654 nm and 540 nm (I654/I540) and the intensity ratio of 654 nm and 1550 nm (I654/I1550) were calculated (Fig. 3A). Both the I654/I540 and the I654/I1550 were positively linearly correlated with the concentration of ·OH. The slope of the intensity ratio and ·OH concentration (1.16 for I654/I540 and 18.24 for I654/I1550) and the relative intensity change (3.46 for I654/I540 and 35.12 for I654/I1550) at 1550 nm at the concentration of 500 μM were higher, as compared with the visible reference at 540 nm (Fig. 3B), thus suggesting that 1550 nm has higher sensitivity as a reference for the detection of ·OH. We then attempted to explain the


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mechanism underlying this result. The absorbance of csEr-Cy overlapped with the UCL emission peaks of csEr at 654 nm, thus causing inner filter effect (IFE) and quenching the emission, whereas the SWIR emission at 1550 nm was not influenced (Fig. 3C). In addition, to provide insights into the frontier molecular orbital distribution and to propose a mechanism of energy transfer between csEr and cypate, we performed density functional theory (DFT) calculations with the B3LYP exchange-correlation function by using a 6-31g basis set (Fig. 3D). The structure of cypate is optimized and the frontier molecular orbital is obtained. According to the corresponding distributions of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of cypate, Förster resonance energy transfer (FRET) generation can occur between csEr and cypate, because of the higher 4F

9/2

of Er3+ than the LUMO of cypate, thus, causing the quenching of green UCL

emission at 540 nm (2H11/2→4I15/2 and 4S3/2→4I15/2 transition of Er3+) and more effective quenching of the red UCL emission at 654 nm (4F9/2→4I15/2 of Er3+). However, there was no effect on the SWIR emission at 1550 nm because the LUMO of cypate was higher than the 4I13/2 of Er3+. After reaction with ·OH, IFE and FRET cannot occur, and the UCL emission at 540 nm and 654 nm recover. Given the influences of IFE and FRET, the SWIR emission is more feasible as a reference than visible UCL emission, and interference-free detection can be achieved by using SWIR as a reference, which may hopefully improve the sensitivity of detection. To investigate the influence of 1550 nm penetration on csEr, slices of mouse skin was pasted on the wall of a centrifugal tube, and bright field, UCL, and SWIR images 9 ACS Paragon Plus Environment


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were taken (Fig. 3E). The intensity of 540 nm, 654 nm, and 1550 nm (I540, I654, and I1550) before and after the skin slices were pasted on the wall of the tube were calculated, and the influence of penetration was determined according to the intensity ratios (Iafter/Ibefore) of the intensity values with and without skin (Fig. 3F). The Iafter/Ibefore of 1550 nm was 1.013, and that at 540 nm was 2.681, thus, indicating that the skin slices significantly influenced the UCL image, whereas little change was observed in the SWIR image. These results further demonstrated that using SWIR luminescence as a reference is better than using visible luminescence, because of the low influence of penetration. Therefore, SWIR luminescence as reference have the advantages of no interfere, high sensitivity, and deep penetration.

3.4. Diagnosis of arthritis in vivo through ratiometric imaging csEr-Cy performed well in the detection of ·OH by using SWIR as a reference; therefore, we further studied the diagnosis of arthritis through the detection of ·OH in vivo through ratiometric imaging. First, the toxicity of csEr-Cy was studied in vitro and

in

vivo.

The

cytotoxicity

of

csEr-Cy

was

studied

with

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide (MTT) assays. A high cellular viability of ~97% was calculated even at a high concentration of csEr-Cy (10 mg mL-1) within 48 h, thus, suggesting that csEr-Cy does not significantly influence CCC-HEL-1 cells (Fig. S8). The MTT assay results demonstrated the low cytotoxicity of csEr-Cy. Moreover, no mouse death occurred, nor obvious abnormal behavior was


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observed after the injection of csEr-Cy. The above results demonstrate that csEr-Cy has low toxicity to mice at the tested dose. A bioimaging system was then designed for ·OH detection by csEr-Cy in vivo (Scheme 1). To achieve the diagnosis of arthritis in vivo through ·OH detection, we established an arthritis model in mice which right leg had mild arthritis, whereas the left leg was normal and was used as a control. As shown in bright-field images, we observed that slight swelling appeared on the right leg, thus, suggesting that the arthritis model was successfully established (Fig. 4D, G). csEr-Cy nanoprobe was then used to monitor the ·OH in the arthritic area. After injection with csEr-Cy solution (5 mg mL-1, 50μL), we observed the map of the ·OH distribution through UCL imaging, SWIR imaging and ratiometric imaging (Fig. 4A-C, H). The ·OH concentration in the arthritic area was higher than that in the normal area. The results further indicated that csEr-Cy nanoprobes were scarcely affected by the autoluminescence signals and light scattering in the arthritic area. Using the SWIR signal as a reference, we calculated the ratio of red and SWIR emission (R/S) in the mouse legs to assess ·OH ratiometric imaging feasibility. The R/S ratio in the legs with arthritis (4.1430 ± 0.2791) was significantly higher than that in the control legs (1.0000 ± 0.1677) (Fig. 4I), results in good agreement with the ratiometric imaging in vivo. The above results demonstrated the feasibility of using csEr-Cy in the diagnosis of arthritis through ratiometric imaging in vivo. Therefore, the csEr-Cy nanoprobe is capable of monitoring ·OH accumulation in vivo with the SWIR signal used as a reference. For comparison, we also investigated the ratiometric ·OH detection and 11 ACS Paragon Plus Environment


arthritis diagnosis effects by using 540 nm emission as a reference. The ratiometric imaging shows the position of arthritis area (Fig. 4E). The ratio of red emission and green emission (R/G) in the legs with arthritis was calculated (1.3758 ± 0.0377) (Fig. 4F). However, both the ratiometric imaging and ratio of R/G were not as significant as those of R/S. The results suggested that interfere-free detection of ·OH and a high sensitivity arthritis imaging diagnosis can be achieved by using the SWIR signal as a reference, which is better than UCL reference.

4. Conclusion In this work, a novel cypate-modified core-shell rare earth-based nanoprobe was rationally designed for the detection of ·OH and the diagnosis of arthritis. Owing to the reaction between cypate and ·OH, the emission of Er3+ at 654 nm is quenched, and the SWIR emission at 1550 nm is not affected, and therefore, was used as a reference for interference-free ratiometric detection. The slope of the intensity ratio and ·OH concentration, and the relative intensity change at 1550 nm were higher, as compared with the visible reference at 540 nm; thus, suggesting that 1550 nm has high sensitivity as a reference for the detection of ·OH (limit of detection = 4.20 μM). Besides, the penetration depth of 1550 nm is also better than that of 540 nm. UV-vis-NIR spectrum and DFT calculations showed that cypate influences the intensity of 540 nm and 654 nm through IFE and FRET, but does not affect that at 1550 nm. In addition, the csEr-Cy nanoprobe was successfully used in mice for the


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diagnosis of arthritis in vivo. This study provides a novel strategy for high sensitivity detection in vivo; thus, demonstrating the possibility of using SWIR signals as a reference, since they performed better than normal references in visible regions.

Acknowledgement The authors thank the funding of Beijing Talent Foundation Outstanding Young Individual Project (2015000026833ZK02), Beijing Municipal Education Commission Outstanding Young Individual Project (CIT&TCD201904082), The Joint Foundation Program of Beijing Municipal Natural Science Foundation and Beijing Municipal Education Commission (KZ201810028045), Capacity Building for Sci-Tech Innovation-Fundamental Scientific Research Funds (19530050179), Project of High-level Teachers in Beijing Municipal Universities in the Period of 13th Five-year Plan (IDHT20180517), Project of Construction of Scientific Research Base by the Beijing Municipal Education Commission, Yanjing Young Scholar Program of Capital Normal University, and Youth innovative research team of Capital Normal University.

The Supporting Information is available free of charge on the ACS Publications website. Detailed experiment section; Size distribution of NaErF4 and csEr; FTIR and UV-vis-NIR spectra of csEr-Cy; FTIR of csEr and free cypate; Digital images of 13 ACS Paragon Plus Environment


NaErF4, csEr, and csEr-Cy; Intensity changes at 540 nm and 1550 nm; MTT assay; Comparison of csEr-Cy and Previously Reported Nanoprobes for Hydroxyl Radical Detection.

Competing financial interests The authors declare no competing financial interest.

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(17) Shariatdoust, M. S.; Frencken, A. L.; Khademi, A.; Alizadehkhaledi, A.; van Veggel, F. C. J. M.; Gordon, R. ACS Photonics 2018, 5, 3507-3512. (18) Shi, Z. L.; Duan, Y.; Zhu, X. J.; Wang, Q. W.; Li, D. D.; Hu, K.; Feng, W.; Li, F. Y.; Xu, C. X. Nanotechnology 2018, 29, 094001. (19) Guo, Q.; Liu, Y.; Jia, Q.; Zhang, G.; Fan, H.; Liu, L.; Zhou, J. Anal. Chem. 2017, 89, 4986-4993. (20) Liu, Y.; Jia, Q.; Guo, Q.; Jiang, A.; Zhou, J. Anal. Chem. 2017, 89, 12299-12305. (21) Wang, N.; Yu, X.; Zhang, K.; Mirkin, C. A.; Li, J. J. Am. Chem. Soc. 2017, 139, 12354-12357. (22) Guo, M.; Huang, J.; Deng, Y.; Shen, H.; Ma, Y.; Zhang, M.; Zhu, A.; Li, Y.; Hui, H.; Wang, Y.; Yang, X.; Zhang, Z.; Chen, H. Adv. Funct. Mater. 2015, 25, 59-67. (23) Wan, H.; Ma, H.; Zhu, S.; Wang, F.; Tian, Y.; Ma, R.; Yang, Q.; Hu, Z.; Zhu, T.; Wang, W.; Ma, Z.; Zhang, M.; Zhong, Y.; Sun, H.; Liang, Y.; Dai, H. Adv. Funct. Mater. 2018, 28, 1804956. (24) Cui, J. B.; Jiang, R.; Guo, C.; Bai, X. L.; Xu, S. Y.; Wang, L. Y. J. Am. Chem. Soc. 2018, 140, 5890-5894. (25) Wang, S. F.; Fan, Y.; Li, D. D.; Sun, C. X.; Lei, Z. H.; Lu, L. F.; Wang, T.; Zhang, F. Nat. Commun. 2019, 10, 1058. (26) Li, D.; Wang, S.; Lei, Z.; Sun, C.; El-Toni, A. M.; Alhoshan, M. S.; Fan, Y.; Zhang, F. Anal. Chem. 2019, 4771-4779. (27) Tang, Y.; Li, Y.; Wang, Z.; Pei, F.; Hu, X.; Ji, Y.; Li, X.; Zhao, H.; Hu, W.; Lu, X.; Fan, Q.; Huang, W. Chem. Commun. 2019, 55, 27-30. (28) Xu, J. T.; Gulzar, A.; Yang, P. P.; Bi, H. T.; Yang, D.; Gai, S. L.; He, F.; Lin, J.; Xing, B. G.; Jin, D. Y. Coord. Chem. Rev. 2019, 381, 104-134. (29) Yang, D. P.; Cao, C.; Feng, W.; Huang, C. H.; Li, F. Y. J. Rare Earths 2018, 36, 113-118. (30) He, S. Q.; Song, J.; Qu, J. L.; Cheng, Z. Chem. Soc. Rev. 2018, 47, 4258-4278. (31) Miao, Q. Q.; Pu, K. Y. Adv. Mater. 2018, 30, 1801778. (32) Xu, M.; Zou, X. M.; Su, Q. Q.; Yuan, W.; Cao, C.; Wang, Q. H.; Zhu, X. J.; Feng, W.; Li, F. Y. Nat. Commun. 2018, 9, 2698. (33) Wang, S.; Liu, L.; Fan, Y.; El-Toni, A. M.; Alhoshan, M. S.; Li, D.; Zhang, F. Nano lett. 2019, 2418-2427. (34) Shao, W.; Chen, G. Y.; Kuzmin, A.; Kutscher, H. L.; Pliss, A.; Ohulchanskyy, T. Y.; Prasad, P. N. J. Am. Chem. Soc. 2016, 138, 16192-16195. (35) Dong, A. G.; Ye, X. C.; Chen, J.; Kang, Y. J.; Gordon, T.; Kikkawa, J. M.; Murray, C. B. J. Am. Chem. Soc. 2011, 133, 998-1006. (36) Zhao, Q.; Zhang, R.; Ye, D.; Zhang, S.; Chen, H.; Kong, J. ACS Applied Materials & Interfaces 2017, 9, 2052-2058. (37) Zhuang, M.; Ding, C.; Zhu, A.; Tian, Y. Analytical Chemistry 2014, 86, 1829-1836.



Scheme 1. Schematic illustration of an interfere-free ·OH detection mechanism of csEr-Cy nanoprobe and its application in vivo.


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Figure 1. Synthesis and characteristic of csEr-Cy nanoprobes. TEM image of NaErF4 (A) and core-shell NaErF4@NaLuF4 (csEr) (B). Scale Bar: 100 nm. (C) HR-TEM image of csEr. Scale Bar: 2 nm. Insert: corresponding fast Föurier transformation diffractogram. (D) XRD pattern of NaErF4 and csEr. (E) EDXA spectrum of csEr. (F) UV-vis-NIR spectra of csEr-Cy before and after the addition of ·OH.



Figure 2. Ratiometric luminescence detection of ·OH (A) UCL spectra of csEr-Cy reacted with different concentration of ·OH (from 0 to 500μM). Insert: the UCL images of csEr-Cy before/after react with ·OH. (B) SWIR spectra of csEr-Cy reacted with different concentration of ·OH (from 0 to 500μM). (C) UV-vis-NIR spectrum of csEr-Cy reacted with ·OH within 20 min. (D) UV-vis-NIR response of csEr-Cy solution in the aqueous containing different final concentration of ions and ROS: 5 mM for Na+, K+, Ca2+, Mn2+, Fe2+, Zn2+, Cl-, CO32-, SO42-, NO3-, PO43-, H2O2, and ClO-. ·OH (5μM) was used for reference.


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Figure 3. Sensitivity studies utilizing various references. (A) Linear relationship between ·OH concentrations and I654/I540 and I654/I1550 in the range from 0 to 500 μM. (B) Slope obtained from Fig. 3A, and relative intensity change obtained from the UCL and SWIR spectra from Fig. 2A, B. (C) UV-vis-NIR absorbance and SWIR luminescence spectra of csEr-Cy. (D) Proposed mechanism of energy transfer between csEr and cypate calculated by DFT. (E) The bright field, UCL and SWIR images of csEr before and after skin slices pasted on the wall of the tube. The signals were collected at the wavelength of 654 nm, 540 nm, and 1550 nm, respectively. (F) The intensity ratio of green (I540) and SWIR (I1550) emissions before and after skin slices pasted on the wall of the tube (Iafter/Ibefore) obtained from Fig. 3E.



Figure 4. Diagnose of arthritis in vivo by ratiometric imaging. The UCL images at 540 nm (A) and 654 nm (B), and (C) the SWIR image at 1550 nm for detection of ·OH based on csEr-Cy in an inflammatory mode. The bright-field image (D) and the ratiometric (R/G) image (E) for ratiometric detection of ·OH based on csEr-Cy in an inflammatory mode. (F) R/G signal obtained from UCL images of normal or inflamed mice legs from Fig. 4D, E. The bright-field image (G) and the ratiometric (R/S) image (H) for ratiometric detection of ·OH based on csEr-Cy in an inflammatory mode. (I) R/S signal obtained from SWIR images of normal or inflamed mice legs from Fig. 4G, H.


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Interfere-free Detection of Hydroxyl Radical and Arthritis Diagnosis by

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