Modulating the Luminescence of Upconversion Nanoparticles with

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Modulating the Luminescence of Upconversion Nanoparticles with Heavy Metal Ions: A New Strategy for Probe Design Tao Liang, Zhen Li, Dan Song, Lin Shen, Qinggeng Zhuang, and Zhihong Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01963 • Publication Date (Web): 15 Sep 2016 Downloaded from http://pubs.acs.org on September 18, 2016

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Analytical Chemistry

Modulating the Luminescence of Upconversion Nanoparticles with Heavy Metal Ions: A New Strategy for Probe Design Tao Liang,† Zhen Li,† Dan Song,† Lin Shen,† Qinggeng Zhuang,‡ Zhihong Liu*† † Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China. ‡Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH 43210 (USA)

*Corresponding author: Zhihong Liu Fax: 86-27-68754067 Email: [email protected]

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ABSTRACT. Upconversion nanoparticles (UCNPs) are attracting increasing attention in biosensing and imaging. The design of UCNPs-based probes currently relies exclusively on luminescence resonance energy transfer (LRET) principle. The prerequisite spectral overlap in LRET leads to limited flexibility in the probe design, thus hindering the construction and application of upconversion (UC) probes. To change this situation, we herein present a new approach to construct UC probes by using the heavy metal ionsinduced quenching. We reveal that heavy metal ions can quench the upconversion luminescence (UCL) to >95% without the occurrence of spectral overlap. A proof-ofconcept UC probe for biothiols by manipulating Cu2+ as the switch of luminescence exhibits satisfying performance both in vitro and in bioimaging. This is the first report on UC probe utilizing heavy metal ions to govern the read-out signal. The strategy is much simpler than the LRET principle and highly efficient, which provides a new way for the design and application of UCNPs-based probes.

INTRODUCTION Upconversion nanoparticles (UCNPs) have drawn increasing attention in bioassay and bioimaging owing to their features of excitation with near-infrared (NIR) light and anti-Stokes luminescence emission.1-6 Different from traditional fluorophores with Stokes emission, UCNPs with anti-stokes emission convert two or more low-energy NIR photons into a high-energy photon.7-10 This property confers a number of excellent optical properties on them, such as preclusion of autofluorescence from bio-samples and interference from scattered excitation light, deeper light penetration and lower photodamage.11-15 Among the various UC probes reported, a common principle is the use of luminescence resonance energy transfer (LRET) to regulate the


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emission of UCNPs as the read-out signal. A number of upconversion LRET systems have been developed employing varying materials as energy acceptors, including organic dyes and inorganic nanosized quenchers.16-23 Generally, the emission of UCNPs is depressed by the energy acceptors followed by a target concentration-dependent recovery. Although LRET is undoubtedly an effective way to govern the signal of the energy donor (UCNPs) and to construct fluorescence probes, the prerequisite spectral overlap between the energy donor’s emission and the energy acceptor’s absorption leads to rigorous confines and limited flexibility in the design of probe. It is known that the absorption spectra of most organic dyes are not easily adjustable; meanwhile the emission bands of UCNPs are extremely narrow. Therefore, it is always challenging to design or select an appropriate organic energy acceptor for UCNPs. Some inorganic nanosized quenchers display broader absorption than organic dyes, thus affording some flexibility in the design of LRET-based UC probes. However, the larger size of these nanomaterials may impair the stability and biocompatibility of the probe.24,25 We recently exploited noble metal nanoclusters as energy acceptors of an upconversion nanoprobe, which combined the favorable biocompatibility and flexibility of small-molecule energy acceptors and the high energy-transfer efficiency featured by nanomaterial acceptors.26 Nevertheless, it is rather tricky to find a suitable template to synthesize the nanoclusters with appropriate absorption spectra overlapping exactly with the emission of UCNPs. Thus, an alternative strategy for designing UC probes with no need of matching spectra is highly desired in order to promote the application of UC probes. To meet the above challenges, we are motivated to seek alternative pathways to modulate the luminescence of probe. Heavy metal ions are known as efficient quenchers of fluorophores and phosphors through diverse mechanisms.27-37 The quenching of UCL by free heavy metal ions in



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solutions was reported by Wolfbeis et al.,38 but no follow-up research was seen to pave way for its applications in probe design. In this work, we reveal that heavy metal ions such as Cu2+ and Fe3+, at relatively lower concentrations, can cause significant quenching of UCL to degrees of >95% by anchoring them onto the surface of UCNPs. On this basis, we propose a new strategy for designing UC probe by manipulating these species as the switch of signal. It is an uncomplicated and more straightforward design since the prerequisite spectral overlap between the donor-acceptor pair in LRET systems is unnecessary for this strategy.39,40 In addition, the simple structure and small size of quenchers endow the UC probes with high flexibility and biocompatibility, which is favorable for application in biological samples. A proof-of-concept UC probe for biothiols, which play crucial roles in maintaining the intracellular redox homeostasis, is constructed using Cu2+ as the quencher. The quencher is anchored to the surface of UCNPs with a small ligand DPEA (N,N-di-(2-picoly)ethylenediamine) which coordinates with Cu2+.41,42 The UCL intensity is recovered upon the removal of Cu2+ by biothiols, which enables both the quantification in vitro and monitoring of the biothiols level in living cells and deep tissues. The strategy shown here also has good extendibility, i.e., it is possible to use a variety of heavy metal ions to modulate UCL of probes for various targets, since there are a considerable number of candidates of metal ions with luminescence quenching ability.43,44 Therefore, it can be a useful alternative to LRET principle and provide an effective new choice for designing UC probes.

EXPERIMENTAL SECTION Synthesis of Oleate-covered Sandwich-structure Upconversion Nanoparticles. Seed-mediated growth approach was used to prepare sandwich-structure upconversion


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nanoparticles (SWUCNPs) capped with oleate, as described previously.23,45 The matrix was NaYF4, and Yb3+ and Tm3+ were doped as the sensitizer and the activator in the middle layer (inner shell) of the sandwich-structure nanoparticles. Oleic acid/1octadecene (equal volume) was used as the solvent. To 10 mL of solvent, Y(oleate)3 (1 mmol) and NaF (20 mmol) were added. The solution was degassed in vacuum at 110 °C for 1 h in a three-neck flask, then kept at 320 °C for core growth in argon atmosphere. An aliquot of 4 mL of the reaction was acquired 75 min later and saved for characterization. Next, 0.4 mmol of Ln(oleate)3 (Y3+:Yb3+:Tm3+= 79.8:20:0.2) dissolved in 8 mL of solvent was added to the reaction for the growth of the middle layer. A second aliquot of 6 mL was taken 20 min after the addition for characterization. Finally, 0.4 mmol of Y(oleate)3 dissolved in 8 mL of solvent was added for the growth of the outer layer. The reaction was terminated 20 min after the addition by cooling to room temperature. The first and second intermediate aliquots were the cores and the core-shell particles, respectively, and the final product was SWUCNPs (NaYF4@NaYF4:Yb,Tm@NaYF4), all were capped by oleate. Nanoparticles were precipitated from the solutions by adding equal volumes of EtOH followed by centrifugation, then washed six times with 1:1 hexane/EtOH. Coating SWUCNPs with Poly (acrylic acid). Oleate-capped SWUCNPs were treated with HCl to remove the oleate ligand.46,47 60 mg of oleate-capped SWUCNPs were added to 30 mL of HCl solution in EtOH at pH 1.0, then sonicated for an hour. Ligand-free SWUCNPs were harvested by centrifugation, and then rinsed with an HCl/EtOH solution at pH 4.0 once, and with ethanol and ultrapure water several times. The product was redispersed in ultrapure water for further use. 40 mg of ligand-free SWUCNPs were added to 800 mg of poly (acrylic acid) (PAA) dissolved in 20 mL of ultrapure water. The reaction was stirred vigorously at room temperature



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for 24 h. Yielded PAA-coated SWUCNPs were precipitated by centrifugation, washed three times with EtOH and three times with ultrapure water. Synthesis of DPEA. N,N-di-(2-picoly)ethylenediamine (DPEA) was synthesized according to the procedures described in a previous report.48 Preparation of SWUCNPs-DPEA-Cu(II) probe. Carboxyl of PAA-coated SWUCNPs (1 mg) were activated with EDC (0.5 mg) and NHS (1 mg) in 1 mL of MES (pH=5.5) for 15 min, then reacted with 400 nmol of DPEA overnight at room temperature for conjugation. Yielded DPEAconjugated SWUCNPs were precipitated by centrifugation, then washed three times with ultrapure water and redispersed in 1 mL of HEPES buffer (10 mM, pH 7.2). Varying amounts of Cu(NO3)2 solution were added and incubated at 37 °C for 3 h. The products were collected by centrifugation and washed three times with ultrapure water and redispersed in HEPES buffer to a final concentration of 1 mg/mL (nanoparticle) for further use. Quenching of UCL by Fe3+. Bared SWUCNPs (1 mg/mL) were incubated with varying amounts of hemin (0~100 µM) in HEPES buffer (10 mM, pH=7.2) overnight at room temperature. The products were collected by centrifugation and washed three times with ultrapure water, then re-dispersed in ultrapure water for UCL measurement. Protoporphyrin IX (PPIX) containing no iron was tested as the control with the same method. In vitro Assay of GSH with SWUCNPs-DPEA-Cu(II). In HEPES buffer (10 mM, pH 7.2), 0.05 mg/mL SWUCNPs-DPEA-Cu(II) was mixed with various concentrations of GSH (0.01~500 µM) and incubated at 37 °C for 40 min. UCL was then measured at 478 nm, excited with continuous-wave laser at 980 nm.


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Selectivity of the SWUCNPs-DPEA-Cu(II) Probe. In the control group, 0.05 mg/mL SWUCNPs-DPEA-Cu(II) was mixed with 10 µM GSH and incubated for 40 min. In other groups, the SWUCNPs-DPEA-Cu(II) solutions were individually incubated with 10 µM interfering substances. Then UCL was measured at 478 nm, excited with continuous-wave laser at 980 nm. Stability of SWUCNPs-DPEA-Cu(II) Probe. To study the photostability, the SWUCNPs-DPEA-Cu(II) composite (0.05 mg/mL) were incubated at 37 °C and UCL was recorded at an interval of 10 min for 1.5 h. Buffers with different pH were prepared by using phosphate salts. UCL signals of 0.05 mg/mL m SWUCNPs-DPEA-Cu(II) in these buffers were measured to examine the pH dependence. Cell Culture. HeLa cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 mg/L streptomycin and 100 U/mL penicillin, in a 24-well microplate with cover glasses for 48 h at 37 °C under 5% CO2. Cytotoxicity. The cytotoxicity of the probe was examined by means of MTT assay with HeLa cells. After cultivated, the cells were incubated in fresh DMEM medium with various concentrations of SWUCNPs-DPEA-Cu(II) (0-0.6 mg/mL) added for 24 h. Six replicates were tested at each concentration. After the incubation with SWUCNPs-DPEA-Cu(II), cells were incubated with 1 mg/mL MTT reagent for 4 h. Produced formazan was dissolved in DMSO. The absorbance at 490 nm was determined. Cell viability (%) was calculated as (mean Abs. of SWUCNPs-treated wells/mean Abs. of control wells)×100%. Biothiol imaging in HeLa cells. HeLa cells were rinsed three times with PBS, then incubated in 0.3 mg/mL SWUCNPs-DPEA-Cu(II) for 1.5 h under 5% CO2 at 37 °C, and rinsed three times



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again. In the positive control group, cells were pretreated with 1 mM of GSH for 1 h at 37 °C prior to the addition of probes. In two negative control groups, cells were pretreated with 0.5 and 1 mM of N-methylmaleimide (NMM). In another group, cells were incubated with 100 µM GSH after treating with 1 mM NMM. Images were acquired using a Nikon Ni-E Microscope equipped with 980 nm laser for excitation. Biothiol imaging in mice liver. Kunming mice (approximately 30 g) were pretreated with 200 µL of para-acetylaminophenol (APAP) solution (0, 10, 20 or 40 mg/100 g body weight, dissolved in pH 7.4 HEPES) by intravenously (i.v.) injection. 30 min later, mice were intraperitoneally (i.p.) injected with SWUCNPs-DPEA-Cu(II) (3 mg/100 g body weight in 300 µL physiological saline). Livers were harvested 1 h after the injection of SWUCNPs-DPEA-Cu(II), then sliced and observed under microscope. Control group 1 had the SWUCNPs-DPEA-Cu(II) solution displaced with blank saline. Control group 2 was i.v. injected with 100 µL of α-lipoic acid (1 mg/100 g and 2 mg/100 g body weight, 0.05 M NaOH, dissolved in physiological saline) 1 h before the injection of the APAP (40 mg/100 g).

RESULTS AND DISCUSSION Quenching of UCL by Heavy Metal Ions. We first chose Cu2+ as a representative to explore the quenching effect of heavy metal ions to the UCL. Sandwich-structure UCNPs (SWUCNPs) were used as the luminophore for its higher efficiency of quenching as proved in our previous work.49 DPEA is a well ligand for Cu2+ and usually used to recognize or anchor Cu2+. In a previous work,42 a pulse voltammetry method for the detection of Cu2+ and cysteine with DPEA as the recognition unit for Cu2+ was reported,


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in which the change of electrochemical reduction peak of Cu2+ was employed as signal output. In our present work, we used DPEA to anchor Cu2+ on the surface of UCNPs (upconversion nanoparticles), with the aim of constructing an optical probe through the modulation

of

the

luminescence

of

UCNPs

by

Cu2+.

SWUCNPs

(NaYF4@NaYF4:Yb,Tm@NaYF4) were prepared through a layer-by-layer seed-mediated shell growth pathway, and the size evolution of the products was characterized by transmission electron microscopy (TEM) (Figure S1a-c). The crystalline phase was verified with selected-area electron diffraction (SAED) image, which showed typical hexagonal-phase NaYF4 lattice and agreed with the results of powder X-ray diffraction (XRD) (Figure S1d-f). To assemble DPEA, the oleate ligands on the particle surface were replaced by poly(acrylic acid) (PAA). The oleate ligands were firstly removed by acid treatment to expose lanthanide ions which can coordinate with PAA carboxyl. Functionalization with PAA was confirmed by Fourier transform infrared (FTIR) analysis and zeta potential measurements (Figure S2, S3). The XRD patterns displayed that the crystalline phase of the nanoparticles was not affected by ligand replacement (Figure S4a). The DPEA molecules were then covalently linked via the formation of amide bond between the amino group in DPEA and the PAA carboxyl group on the particle surface, and successful conjugation was confirmed by ζ potential determination and UV-Vis spectroscopy. After the conjugation of DPEA, the ζ potential of PAA-SWUCNPs shifted from -10.5 mV to -7.34 mV (Figure S3) since DPEA is positively charged. The characteristic absorption of DPEA at 260 nm was also observed in the UV-Vis spectrum of the nanoparticles (Figure S5a).



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Figure 1. (a) Emission of SWUCNPs-DPEA (0.05 mg/mL) quenched by different concentrations (0-20 µM) of Cu2+. (b) Relative luminescence intensity (F/F0, in which F and F0 represent the UCL emission intensity in the presence and the absence of Cu2+, respectively) of SWUCNPsDPEA at three emission wavelengths after reacting with different concentrations of Cu2+. Subsequently, we investigated the quenching of UCL by Cu2+ which was coordinated with the DPA (dipicolylamine) moiety to form SWUCNPs-DPEA-Cu(II) complex. The successful binding of Cu2+ on particles was verified by several means. The absorption at 260 nm was slightly blue-shifted and increased in intensity, which implies the formation of DPEA-Cu(II) complex (Figure S5a). The X-ray photoelectron spectroscopy (XPS) survey spectrum of the SWUCNPs-DPEA-Cu(II) composite also showed signals that can be assigned to Y 3d orbitals (158 and 162 eV) and to Cu 2p orbitals (933 and 952 eV), as another evidence confirming that Cu2+ ions were bound on the surface of SWUCNPs (Figure S6). As seen in Figure 1a, the UCL intensities at three emission wavelengths of SWUCNPs were negatively correlated with the amount of added Cu2+, and ultimately all of the emissions were almost completely quenched (Figure 1b). This result is basically consistent with that reported by Wolfbeis et al.,38 except that the metal ions were anchored onto the surface of particles in our work rather than diffused in the solution, which leads to higher quenching efficiency at much lower amount and also enables the fabrication of UC probe. Notably, this is an outstanding quenching efficiency as compared to


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those LRET-based quenching of UCNPs, indicating that the use of heavy metal ions could be a very simple yet effective approach. It is worth pointing out that the upconversion emission process of Ln3+ is in principle different from the downconversion Ln3+ complexes, since they are based upon different sensitization processes, i.e., with different antennas (organic ligands versus lanthanide ions like Yb3+).7,50 Moreover, the involvement of multi-photon process and multiple energy converting pathways between the sensitizer and emitter, as well as the crystal field effect of host matrix make the UCL even more sophisticated.7 Accordingly, the quenching of the UCL can also be more complicated than that of downconversion Ln3+ complexes.38 The lifetimes of emissions in water at 478 nm and 650 nm were 1.00 and 0.98 ms, which are comparable with those in literatures,49,51 and decreased to 0.75 and 0.48 ms after addition of Cu2+, respectively (Figure S7a,b), indicating the existence of non-static quenching.26,49,52 Our analysis on TEM images (Figure S4b-d) and hydrodynamic diameter (Table S1) shows that Cu2+ did not cause aggregation of SWUCNPs-DPEA, thus ruling out the possibility of aggregation-induced quenching. In those Ln3+ complexes or Ln3+ doped downconversion materials, resonant energy transfer is an important factor accounting for the quenching because the absorption of the quenchers overlaps with the emission of either the emitter or the sensitizer.23,53 But in our case, such spectral overlap did not exist. As shown in Figure S5b, neither DPEA nor DPEA-Cu(II) exhibited any absorption in the range of the donor’s emission, which excludes the possibility of this process. In addition, to rule out the attenuation of excitation light, the absorption of the quencher in NIR range was measured. As shown in Figure S8, the absorption of both Cu2+ and DPEA-Cu(II) at 980 nm were extremely weak with the molar absorption coefficients of only 5.0 and 47 L mol-1 cm-1, respectively, which indicates that the quenching is not by inner filter effect either.54



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Figure 2. (a) Emission of upconversion nanoparticles quenched by different concentrations (0-5 µM) of Fe(III) caged in hemin. (b) Luminescence quenching efficiency (F/F0) of SWUCNPs at three emission wavelength in presence of different concentrations of hemin. To shed more light on the above interpretations, we then tested another kind of heavy metal ion, Fe3+. To anchor Fe3+ to UCNPs, we used hemin, an iron-containing porphyrin compound with two carboxyl groups that can coordinate with the lanthanide ions on the surface of UCNPs. In agreement with our expectation, all three tested UCL emissions were quenched along with the increase of hemin concentration and finally to a degree of >95% (Figure 2a, 2b). To illustrate that the quenching ability of hemin can be mainly attributed to the metal, we tested protoporphyrin IX (PPIX), a porphyrin without iron, as a control. At comparable levels of spectral overlap integral, the quenching of UCL by PPIX was much lower than by hemin (Figure S9), which confirms the role of Fe3+. Similar to the case of Cu2+, neither Fe3+ nor hemin shows any absorption at around 980 nm (Figure S10). To illustrate the versatility of UCL quenching by metal ions, we examined another kind of UCNP, NaYF4@NaYF4:Yb,Er@NaYF4, which emits at different wavelengths from the above Tm-doped UCNP. We found the emission of the Er-doped UCNPs could also be efficiently quenched by both Cu2+ and Fe3+ (Figure S11), which again verifies that the quenching is not depended on spectral overlap.


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As mentioned above, the anchorage of the metal ions onto UCNPs is essential for the construction of probe, because it not only provides a handle for manipulation (a targeting site) but also affords improved quenching efficiency. Our control experiments showed that the ions anchored on the nanoparticles exhibited much stronger quenching ability than that diffused freely in solutions (Figure S12). In their early studies, Wolfbeis and coworkers assigned the quenching to a collision-mediated dynamic quenching at lower concentration of metal ions, and a mixing dynamic and static quenching at higher concentrations.38 The concentrations of metal ions we used were orders of magnitude lower than that of the literature work. Our results also support the mixed mechanism which can find evidences from the viscosity dependent UCL quenching and the Stern-Volmer plot. It was observed that the rate of quenching decreased along with the increase of the viscosity of solvent (Figure S13), indicating the presence of collision-based quenching.55 Linking the quenchers to the surface of UCNPs through a flexible linker enhances the rate of collision, which could be the reason that we gained higher quenching degrees at lower concentrations of metal ions. Meanwhile, the Stern-Volmer plot exhibited an upward curvature concaving toward the y-axis, which is the characteristic feature of the combination of dynamic and static quenching (Figure S14).

18,56

We must acknowledge that, because of the high

complexity of upconversion luminescence and the fact that we did not focus on the physicochemical investigation in this work, the exact mechanism underlying the quenching is not yet absolutely clear. However, we can speculate from all our observations that the quenching of UCL by these heavy metal ions is essentially not a result of the resonant energy-transfer process.56,57 Therefore, we can expect an effective new way for the construction of UC probes as an alternative to the currently adopted LRET principle.



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Scheme 1. Principle of the upconversion nanoprobe for biothiols with Cu2+ as the switch of luminescence. The Upconversion Probe for Total Biothiols. We thereafter built a heavy metal ionsbased UC probe for total biothiols taking Cu2+ as an example of UCL modulator, as briefly depicted in Scheme 1. With increasing amounts of GSH added, the UCL of the probe was gradually recovered in a [GSH]-dependent manner (Figure 3a). The relative luminescence intensity of the probe ((F-F0)/F0, in which F and F0 are the UCL intensity in the presence and the absence of GSH, respectively), is linearly correlated with the logarithm of the GSH concentration in the range of 0.01-500 µM (Figure 3b). The relative standard deviation of three independent detections was at the level of 5%, and the detection limit was 7.2 nM, calculated as three times of standard deviation of the blank signal based on 11 individual detections. This observation was explained with the removal of Cu2+ from the DPEA-Cu(II) complex when GSH bonded to Cu2+ centre and precipitated it, as characterized by XPS (Figure S15, S16). This UC probe exhibited satisfying stability and reproducibility, as shown by the quite low deviations in Figure 3b. The response of SWUCNPs-DPEA-Cu(II) probe to GSH as a function of time was examined (Figure S17), which reveals that the UCL was recovered to the maximal extent


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within 40 min and reached to a stable plateau. To investigate the specificity of the probe toward biothiols, we tested a series of interfering species including various metal ions, anions, small molecules and proteins. As shown in Figure S18, biothiols were able to induce obvious recovery of luminescence of the probe, but all those thiol-free species caused no signal change. Moreover, the probe exhibited excellent thermodynamic and pH stability (Figure S19). The performances obtained in these tests show that the probe is not only competent for assay of biothiols in vitro, but it also can be suitable for prolonged imaging in biological environments.

Figure 3. (a) UCL emission spectra of SWUCNPs-DPEA-Cu(II) in the presence of various concentrations of GSH. (b) Relative luminescence intensity ((F-F0)/F0) of the probe in response to varying concentrations of GSH, inset: linear relationship between the relative luminescence intensity and logarithm of GSH concentration. Monitoring Biothiols in Living Cells. To evaluate the intracellular usage of the probe, the cytotoxicity of the probe was first examined based on the reduction activity of methyl thiazolyl tetrazolium (MTT) assay. After incubation with different amounts of probe for 24 h at 37 °C, the cell viability of HeLa cells was higher than 90% with the exposure to a dose of up to 0.6 mg/mL, suggesting a low cytotoxicity of the probe (Figure S20). A cell viability of greater than 95% was maintained in the presence of 0.3 mg/mL probe, which



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was the amount we used in the subsequent UCL imaging. The single-cell Z-scanning experiment by confocal microscopy clearly showed that the probes were well distributed within the cells (Figure S21). This result verifies the efficient uptake of the probe into the cytosol of living cells, which could be via endocytosis facilitated by dyneins.58,59 To demonstrate the capability of SWUCNPs-DPEA-Cu(II) for monitoring biothiols in cells, a group of HeLa cells was directly incubated with the probe (0.3 mg/mL) for 1.5 h and analyzed under a fluorescence microscope equipped with a 980 nm laser. Obvious blue UCL was detected in these cells (Figure 4b), as a result of the recognition of intrinsic biothiols which, as known, are at quite high concentration levels. With the aim to investigate whether the probe could respond to the changes of biothiols level in cells, four additional sets of experiment were performed. One group of HeLa cells as positive control was pretreated with 1 mM exogenous GSH for 30 min before incubation with the probe. As seen in Figure 4a, a slight increase of UCL was observed. Another two groups of cells as negative control were pretreated with the thiol-blocking reagent Nmethylmaleimide (NMM)60 at 0.5 and 1 mM for 30 min, respectively, before incubation with the probe. The luminescence of these samples, as expected, was obviously weaker than that of the above two groups, as NMM scavenged the intracellular biothiols. Moreover, the decrease of UCL intensity was corresponding to the amount of NMM (Figure 4c, 4d). Further, to gain a clearer profile of the adjustment of intracellular biothiols, we conducted another test where 100 µM GSH was added into the biothiols depleted (by 1 mM NMM) cells. The UCL in this group was in turn significantly enhanced (compare Figure 4e with 4d). Taken together, these results confirmed the ability of SWUCNPs-DPEA-Cu(II) to monitor intracellular biothiols.


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Figure 4. (a-e) Confocal microscopic images of HeLa cells incubated with SWUCNPs-DPEACu(II) nanoprobe (0.3 mg/mL): (a) cells pretreated with 1 mM GSH for 30 min prior to the addition of the nanoprobe; (b) cells incubated with the nanoprobe only; (c) cells pretreated with 0.5 mM NMM for 30 min prior to the addition of the nanoprobe; (d) cells pretreated with 1 mM NMM for 30 min before incubation with the nanoprobe and (e) cells pretreated with both 1 mM NMM and 100 µM GSH prior to the addition of the nanoprobe. Scale bar: 50 µm. (f) Normalized average UCL intensities in (a-e) relative to (a). Images were acquired at 450-500 nm. Detection of Biothiols in Mice Liver. The results obtained from cell studies verify that the SWUCNPs-DPEA-Cu(II) probe is suitable for detecting biothiols in biological environments. To validate its practical applicability, we used the probe to track the liver biothiol content in a mouse model. It was documented that the overdose of drugs may induce tissue damage. For example, acetaminophen (APAP), a painkiller safe when utilized at therapeutic levels, can cause severe liver damage61 and the depletion of GSH in liver cells with overdose.62 We thus demonstrated the capability of our probe to detect GSH in an animal model of APAP-promoted tissue damage. The mice i.p. injected with



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the probe (3.0 mg/100 g (body weight, same as below)) in 300 µL of physiological saline exhibited clear UCL signal contributed by the intrinsic biothiols in normal mice liver (Figure 5a), which is similar to the result in HeLa cells and demonstrates the successful transportation and accumulation of the probe in the livers of mice. On the contrary, no signal was seen in the group injected with only physiological saline (Figure 5g), due to the negligible autofluorescence of the biological sample illuminated with 980 nm light. Treatment of the mice by i.v. injection with APAP at 10, 20 and 40 mg/100 g, respectively, before the injection of 3.0 mg/100 g probe, resulted in a stepwise decrease in the UCL intensity with increasing amount of APAP (Figure 5b-d). According to previous reports, the APAP-induced decrease in the GSH level of liver can be suppressed by pretreatment with α-lipoic acid (α-LA).63 We therefore designed two more groups of sample to test the protective effect of α-LA in association with alterations in the GSH level of liver tissue. For the mice pretreated with α-LA before the injection of APAP, the UCL intensities were higher than that of the group injected with the same dosage of APAP, and elevated according to the increasing amount of injected α-LA (Compare Figure 5e, 5f with 5d). The UCL intensities of the samples are also quantitatively illustrated and can be clearly compared in Figure 5h. All the above results have demonstrated the capability of the as-constructed SWUCNPs-DPEA-Cu(II) upconversion probe to study the specific physiological processes associated with the fluctuation of biothiol content in tissues.


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Figure 5. (a-g) Upconversion luminescence imaging of mouse liver slice. (a-d) Mice injected with APAP [a: 0 mg/100 g (body weight, same as below), b: 10 mg/100 g, c: 20 mg/100 g, d: 40 mg/100 g] 0.5 h followed by the nanoprobe. (e-f) Mice injected with αLA [e: 1 mg/100 g, f: 2 mg/100 g] 1 h followed by APAP (40 mg/100 g) and the nanoprobe. (g) Mice injected with only physiological saline. Scale bar: 50 µm. (h) Normalized mean UCL intensities in (a-g), relative to (a). Images were acquired at 450500 nm. CONCLUSIONS In summary, a new strategy for designing upconversion probes has been developed by using heavy metal ions-induced quenching of UCNPs. This design strategy is uncomplicated and straightforward without the requirement of spectral overlap or introduction of large-size energy acceptors. Heavy metal ions such as Cu2+ and Fe3+ can cause the quenching of upconversion luminescence to degrees of >95%, which is a pronounced level and contributes to minimized background. The as-constructed probe for biothiols with Cu2+ as the quencher possesses good sensitivity, high stability and biocompatibility. It is able to monitor biothiols in living cells and tissues, thus it can be a useful tool to study the biological events associated with the change of biothiols content. As the first upconversion probe with heavy metal ions as the signal regulator, it



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features simplicity, high efficiency and extendibility, which may open a new way to the design and application of UCNPs-based probe. ASSOCIATED CONTENT Author Information Corresponding Author *Zhihong Liu. E-mail: [email protected]. Phone: 86-27-8721-7886. Fax: 86-27-6875-4067. Notes The authors declared no completing financial interest. Acknowledgement This work has been financially supported by the National Natural Science Foundation of China (No. 21375098, 21575109). The authors are grateful to Prof. Xueyuan Chen and Dr. Datao Tu for their help with lifetime measurements. Supporting Information Available The XRD, TEM, FTIR characterization of SWUCNPs, the results of ζ potential measurements, the stability and cell viability of probe. This material is available free of charge via the Internet at http://pubs.acs.org./ REFERENCES (1) Chen, G.; Ågren, H.; Ohulchanskyy, T. Y.; Prasad, P. N. Chem. Soc. Rev. 2015, 44, 16801713.


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Modulating the Luminescence of Upconversion Nanoparticles with

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