Breaking Through the Signal-to-Background Limit of Upconversion

(UC) probes which is the limited signal-to-background ratio (SBR). Since UC nanoprobes ... affording “off-on” signals, almost all UC nanoprobes ar...
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Article Cite This: J. Am. Chem. Soc. 2018, 140, 14696−14703

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Breaking Through the Signal-to-Background Limit of Upconversion Nanoprobes Using a Target-Modulated Sensitizing Switch Tao Liang,† Zhen Li,*,†,‡ Peipei Wang,† Fangzhou Zhao,† Jizhou Liu,† and Zhihong Liu*,† †

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Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), College of Chemistry and Molecular Sciences, Wuhan University, Wuhan 430072, China ‡ Hubei Collaborative Innovation Center for Advanced Organic Chemical Materials, Ministry of Education Key Laboratory for the Synthesis and Application of Organic Functional Molecules and College of Chemistry and Chemical Engineering, Hubei University, Wuhan 430062, China S Supporting Information *

ABSTRACT: Although lanthanide-doped upconversion nanoparticles (UCNPs) have shown great promise in biosensing and bioimaging owing to their excellent photophysical properties, researchers are facing a bottleneck of upconversion (UC) probes which is the limited signal-to-background ratio (SBR). Since UC nanoprobes are basically constructed with a luminescence resonance energy transfer (LRET) process to provide “off−on” signals, the SBR level is principally decided by the luminescence quenching efficiency which is very difficult to further improve through existing approaches. Herein, we put forward a new strategy for fabricating UC nanoprobes using an organic dye as target-modulated sensitizing switch. The dye functions as both the recognition unit for target and a potential sensitizer for upconversion luminescence (UCL). The reaction of the dye with target modulates its photophysical properties, which switches on the sensitization and affords a significantly improved SBR. The idea is validated with a proof-of-concept UC nanoprobe for glutathione (GSH) detection with the SBR of ∼30 (versus a SBR of less than 10 for most current UC nanoprobes). This probe showed good performance in GSH sensing both in vitro and in vivo. Our results indicate that the target-modulated sensitization is a useful new strategy to build UC nanoprobes. And we can reasonably expect that the breakthrough of SBR limit will make UC nanoprobe a more powerful tool in future studies.

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INTRODUCTION Lanthanide-doped upconversion nanoparticles (UCNPs) can emit a UV−vis-NIR photon with relatively higher energy by absorbing two or more lower-energy near-infrared (NIR) photons, which endows them with large light penetration depth in tissues and eliminates autofluorescence from biosamples.1−13 Besides, compared with organic dyes, UCNPs also feature larger anti-Stokes shift, sharper emissions and higher photostability. Therefore, UCNPs are particularly suitable for deep and long-time imaging and tracking in the body.14−17 Indeed, UCNPs-based upconversion (UC) probes have been drawing dramatically increasing attention in biological and medical research in recent years.18−20 Nonetheless, along with the rapid development of UC probes, people soon came to realize an important bottleneck of this kind of nanoprobe, i.e., the very limited signal-tobackground ratio (SBR) after recognizing the targets, which is determinant to the sensitivity of imaging and sensing.21 Such a bottleneck is decided by the design principle of UC nanoprobes. As a type of optical probe affording “off−on” signals, almost all UC nanoprobes are constructed based on the principle of luminescence resonance energy transfer (LRET), in which UCNPs act as energy donors and various energy acceptors are employed to quench the upconversion © 2018 American Chemical Society

luminescence (UCL). Upon the recognition of targets, the LRET process is inhibited or blocked, and accordingly the UCL is recovered. Obviously, in this luminescence off−on procedure, the SBR level is mainly decided by the extent of luminescence quenching, which physically is dependent on the LRET efficiency. Unfortunately, as known, the luminescence quenching degree of UC nanoprobes is restricted and rather hard to improve because of the structural character of UCNPs: these particles are normally of tens of nanometers diameter, with the emitters (rare earth ions) doped in a host crystal. With such a structure, the emitters acting as energy donors are spatially quite “far” away from the exterior energy acceptors. Existing approaches to improve SBR of UC nanoprobes are focused on improving the LRET efficiency, mainly by constraining the emitters, such as Tm3+ and Er3+, and energy acceptors in a shorter distance so that the energy transfer could be more efficient. Reducing the size of UCNPs is an effective way to elevate the ratio of quenchable emitters and improve the luminescence quenching efficiency.22,23 However, the reduction of particle size would inevitably increase the surface-to-volume ratio and induce serious surface quenching, Received: July 11, 2018 Published: October 17, 2018 14696

DOI: 10.1021/jacs.8b07329 J. Am. Chem. Soc. 2018, 140, 14696−14703

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Journal of the American Chemical Society leading to lowered luminescence quantum yield.24 In our previous works, we designed a kind of sandwich-structure UCNPs with the emitters confined in a thin layer near to the particle surface, which improved the LRET efficiency to some extent and meanwhile avoided surface quenching.25,26 Despite these efforts, the quenching efficiency of UCL is still unsatisfying, lower than 90% for most reported probes.27−33 This means the SBR of UC nanoprobe is generally less than 10-fold after recognizing the target, resulting in limited sensitivity. In the past years, during another struggle to improve the light absorption and to enhance the UCL intensity of UCNPs, several groups have proposed an interesting dye-sensitization strategy, in which an organic dye like heptamethine cyanine with strong NIR absorption was introduced as an antenna to circumvent the poor light absorption of rare earth ions and dramatically sensitized the UCL of UCNPs.34−42 In this sensitization system, the dye absorbs NIR photons and then transfers its excited-state energy to the rare earth ions (like Yb3+ or Nd3+) via a nonradiative energy transfer process, due to the spectral match between the emission of the dye and the absorption of these rare earth ions. This gives us a hint to create a new strategy of constructing a UC nanoprobe free of the limitation of UCL quenching efficiency. We consider using an organic dye as a target-modulated sensitizing switch of a UC nanoprobe. In principle, the dye acts as both the recognition unit of the target and a potential antenna. The photophysical properties of this dye can be regulated by the target. Before reacting with the target, the dye is nonemissive and no sensitization occurs. While after reacting with the target, the dye becomes strongly emissive, enabling the nonradiative energy transfer process and causing the sensitization of UCL. In this way, the SBR of the probe no longer depends on the luminescence quenching of UCNPs, which provides the possibility to break through the SBR bottleneck. In the present work, we validated our design strategy by constructing a proofof-concept UC nanoprobe for glutathione (GSH), which is a cancer biomarker and also plays important role in maintaining intracellular redox homeostasis.43,44 We demonstrated the improved performance of this probe both in vitro and in vivo. Our results show that the target-modulated sensitization is a useful new strategy to build UC nanoprobes. We can thus reasonably expect that the breakthrough of SBR limit will make UC nanoprobe a more powerful tool in future studies.

Scheme 1. Schematic Illustration of the Construction of CyUCNPs probe and Sensing of GSH through the TargetModulated Sensitization

(PET) process from cyanine to the nitroazo group.45 It is to say that Cy-GSH itself is nonemissive and cannot cause any sensitization on UCL. On the contrary, the inner filter effect of Cy-GSH on excitation light decreases the intrinsic UCL of CyUCNPs, resulting in negligible background signal before reacting with GSH (vide infra). In the presence of GSH, the nitroazo group can be replaced by GSH to form the product F-SG, which is a strongly emissive molecule because of the removal of the intramolecular PET process. As a consequence, a resonance energy transfer from excited F-SG to Nd3+ occurs and sensitizes the luminescence of UCNPs. Since this sensitization is triggered exclusively by the target GSH and the degree of UCL enhancement can be modulated by GSH concentration, the as-constructed probe will allow quantitative detection of GSH. To realize the assembly of Cy-GSH and UCNPs, two long alkyls chains were tagged to Cy-GSH to match the hydrophobicity of the oleatecoated UCNPs (OA-UCNPs). The amphiphilic polymer PPEG was modified on the surface of OA-UCNPs to form a hydrophobic cavity, in which Cy-GSH can be loaded through hydrophobic interaction.46 Photophysical Property of Cy-GSH and Its Response to GSH. Cy-GSH has a broad and intense absorption peak around 786 nm with a molar absorption coefficient of 1.16 × 105 mol−1·L·cm−1 (Figure S1). The molar absorption coefficient of Cy-GSH at 808 nm (the wavelength of commercial excitation light source) is 4.94 × 104 mol−1·L· cm−1, which is ∼104 times higher than that of Nd3+.40 This is why people can use these dyes as antenna to remarkably sensitize the luminescence of UCNPs. The photoluminescence of Cy-GSH in the absence of GSH is extremely weak due to the existence of PET. After reaction with GSH, the photoluminescence intensity of the reacting product, F-SG, around 810 nm is obviously enhanced with a GSH concentration-dependent manner (Figure S2a), and the emission well matches with the absorption of UCNPs (Figure S2b). These photophysical properties have paved the way to the construction of a sensitization based UC nanoprobe. Fabrication and Characterization of Cy-UCNPs. We adopted the following core−shell structure for UCNPs, NaYF4:20%Yb,2%Er@NaYF4:20% Nd, with the purpose of using the established energy transfer chain: dye → Nd3+ → Yb3+ → Er3+,41 as depicted in Scheme 2. The core−shell nanoparticles were synthesized by a solvothermal method through a seed-mediated shell growth strategy with OA as the capping agent. The transmission electron microcopy (TEM)

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RESULTS AND DISCUSSION Design Principle of the UC nanoprobe Based on Target-Modulated Sensitizing Switch. Following the above-mentioned idea of target-modulated sensitizing switch, we designed a proof-of-concept UC nanoprobe for GSH as illustrated in Scheme 1. The UC nanoprobe, named as CyUCNPs, consists of three parts: the UCNPs, the organic dye Cy-GSH acting as the potential sensitizer as well as GSH recognition unit, and an amphiphilic polymer P-PEG to enhance water solubility of the probe. Heptamethine cyanine was selected as the starting fluorophore to prepare Cy-GSH because its emission spectrum matches well with the absorption of Nd3+,38,41 which makes nonradiative energy transfer from the excited dye to the rare earth ions possible. A nitroazo group was tagged to the heptamethine cyanine scaffold to achieve the target recognition. Another important role of the nitroazo group was to annihilate the emission of heptamethine cyanine via a photoinduced electron transfer 14697

DOI: 10.1021/jacs.8b07329 J. Am. Chem. Soc. 2018, 140, 14696−14703

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Journal of the American Chemical Society

as compared to PEG-UCNPs (Figure S5), ensuring a very low background of the probe. Improved SBR of Cy-UCNPs in the Sensing of GSH. We next investigated the performance of detecting GSH with this UC nanoprobe. With the introduction of a certain amount of GSH, the UCL intensity of the probe gradually increased and reached a stable plateau after about 50 min (Figure S6). Therefore, the Cy-UCNPs probes were incubated with GSH for 1 h in the follow-up experiments to ensure the complete reaction. We also optimized some key factors of the sensing performance. The most important factor, as we consider, would be the thickness of the shell since it acts as a two-edged sword. On one hand, a thicker shell is helpful to resist surface quenching and enhance the UCL intensity; on the other hand, since the energy transfer process is distance dependent, the increase of shell thickness will reduce the energy transfer efficiency both from the dye to Nd3+ and from Nd3+ to Yb3+ (Scheme 2). In our experiments, we prepared four batches of

images (Figure 1a−d) show that the average diameter of the core−shell structure was 41.1 ± 2.2 nm, including the 35.9 ±

Figure 1. Transmission electron microcopy (TEM) images of OAcoated (a) NaYF4:Yb,Er, (b) NaYF4:Yb,Er@NaYF4:Nd. Histograms of the size distribution for nanocrystals of (c) NaYF4:Yb,Er, (d) NaYF4:Yb,Er@NaYF4:Nd. (e) XRD patterns of the core structure NaYF4:Yb,Er and core−shell structure NaYF4:Yb,Er@NaYF4:Nd.

Scheme 2. Schematic Illustration of the Energy Transfer Process from F-SG to UCNPs

1.4 nm core and the 2.6 nm-thick shell. X-ray powder diffraction (XRD) peaks of both the core and core−shell structured UCNPs were consistent with the hexagonal-phase of NaYF4 (JCPDS No. 16-0334) (Figure 1e). To fabricate the Cy-UCNPs probe, Cy-GSH and UCNPs were assembled through hydrophobic−hydrophobic interaction and coated with P-PEG to form water-soluble nanoparticles. Fourier transform infrared (FTIR) spectra and UV− vis absorption spectra were measured to verify the successful construction of the UC nanoprobe. The bands at 1733 and 1647 cm−1 in FTIR spectra of Cy-UCNPs are assigned to C O stretching of the ester groups in Cy-GSH and the amide groups in P-PEG respectively (Figure 2a), suggesting the NaYF4:Yb,Er@NaYF4:Nd with different shell thickness, which were 0.9, 2.6, 4.4, and 5.8 nm, respectively (Figure S7). It was found both the signal-to-background ratio (the UCL enhancement factor F/F0, Figure 3a, Figure S8) and the absolute UCL intensity (Figure S9) were affected by the shell thickness. As anticipated, a moderate thickness (2.6 nm in our system) exhibited the best performance. Another key factor that influences the response to GSH is the amount of Cy-GSH loaded on the nanoprobe. On the same batch of UCNPs (with the shell thickness of 2.6 nm), we loaded different amounts of Cy-GSH which were from 2.1 to 8.5 μg/mg OA-UCNPs (Figure S10), as calculated according to the absorbance of Cy-UCNPs at 786 nm and the molar absorption coefficient of Cy-GSH. As shown in Figure 3b, the UCL enhancement factor was positively correlated with the amount of Cy-GSH assembled on the probe (the corresponding UCL emission spectra are shown in Figure S11). The enhancement factor reached the maximum as the concentration of Cy-GSH was 8.5 μg/mg OA-UCNPs, which was the saturated loading amount of Cy-GSH of this system. Under the above optimal conditions, we recorded the UCL intensity as a function of [GSH] in the range of 0.05−3.0 mM (Figure 3c). To our delight, a maximum SBR, or UCL enhancement factor (F/F0, in which F and F0 represent the UCL intensity in the presence and absence of GSH, respectively) of ∼30 was obtained. This is far beyond the SBR levels of all previously reported UC nanoprobes. A linear correlation between the SBR and the logarithm of GSH concentration was also found

Figure 2. (a) FTIR spectra of Cy-UCNPs, P-PEG, OA-UCNPs and Cy-GSH. (b) UV−vis absorption spectra of Cy-GSH (in DMSO), PEG-UCNPs, and Cy-UCNPs (in 100 mM HEPEs). The concentration of both UCNPs and Cy-UCNPs were 0.05 mg·mL−1.

successful assembly of the three components. In addition, the appearance of the absorption peak at 786 nm of Cy-UCNPs also indicated the anchor of Cy-GSH onto UCNPs (Figure 2b). The ζ potential measurement showed that Cy-UCNPs was positively charged (+29.2 mV, Figure S3), which is favorable for cellular uptake.47 The TEM images also show that the modification of Cy-GSH and P-PEG had no significant effect on the morphology of UCNPs (Figure S4). As mentioned above, the intrinsic luminescence of UCNPs can be suppressed by Cy-GSH through inner filter effect. It was observed that, after the assembly of the components, the UCL intensity of Cy-UCNPs decreased to a maximum rate of 60% 14698

DOI: 10.1021/jacs.8b07329 J. Am. Chem. Soc. 2018, 140, 14696−14703

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Journal of the American Chemical Society

Figure 3. UCL enhancement factor, F/F0, at 545 nm of Cy-UCNPs responding to various concentrations of GSH with (a) different shell-thickness of UCNPs; (b) different amounts of Cy-GSH loaded. (c) UCL enhancement factor of the Cy-UCNPs probe responding to various concentrations of GSH at optimized conditions, and (d) the linear relationship between F/F0 and logarithm of GSH concentration. F0 and F represent UCL intensity in absence and presence of GSH.

Figure 4. (a−e) Confocal microscopic images of HeLa cells incubated with Cy-UCNPs (0.1 mg/mL) for 1.5 h: (a) cells incubated with CyUCNPs only; (b−d) cells pretreated with (b) 1 mM NMM, (c) 2 mM NMM or (d) 5 mM NMM for 0.5 h before addition of the Cy-UCNPs; (e) cells pretreated with 5 mM NMM for 0.5 h and then 1 mM GSH for another 0.5 h before addition of the Cy-UCNPs. (f) Confocal microscopic images of HeLa cells incubated with PEG-UCNPs (0.1 mg/mL) for 1.5 h. (g) Normalized average UCL intensities of (a−f), relative to image a. Images were acquired at 500−600 nm. Scale bar: 10 μm.

under the same conditions as that of GSH. As displayed in Figure S12, the probe showed excellent selectivity against other interferences, including those thiol-containing amino acids such as Cys and Hcy. Furthermore, the UCL intensity hardly changed in the pH range of 5.5−8.0 or during the 60 min incubation at 37 °C (Figure S13a,b), which revealed the high pH stability and thermal stability of this probe. The UCL of

(Figure 3d), which can be the foundation of quantitative detection. The detection limit was 0.6 μM, calculated as 3 times the standard deviation of blank signal based on 7 individual detections, which is comparable with that of previously reported UCNPs-based GSH sensing systems.48 To confirm the specificity of Cy-UCNPs, we tested the response of the probe toward some other ions and molecules 14699

DOI: 10.1021/jacs.8b07329 J. Am. Chem. Soc. 2018, 140, 14696−14703

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Journal of the American Chemical Society

Figure 5. (a−f) UCL images of mouse liver tissues. (a−c) Mice injected with APAP (a: 0, b: 10, c: 40 mg/100 g body weight) 0.5 h before the injection of the Cy-UCNPs. (d,e) Mice pretreated with α-LA (d: 1, e: 2 mg/100 g body weight) for 1 h before injection with APAP (40 mg/100 g body weight) and the Cy-UCNPs. (f) Mice injected with physiological saline. (g) Normalized UCL intensities of images (a−f) (the error bars are acquired with 5 mice each group).

marker), respectively. As shown in Figure S18, Cy-UCNPs randomly distributed in living cells. Thereafter, the Cy-UCNPs probe with satisfying biocompatibility was used to monitor the GSH levels in HeLa cells. Excited by 808 nm laser, obvious UCL was detected in cells after incubation with Cy-UCNPs (0.1 mg/mL) for 1 h (Figure 4a), which can be attributed to the interaction between CyUCNPs and intracellular GSH. In order to verify the ability of this UC nanoprobe to monitor the alteration of GSH level in living cells, four more groups of cells were tested. Among them, three groups of HeLa cells were pretreated with various concentrations of thiol-blocking reagent N-methylmaleimide (NMM)49 for 0.5 h before incubation with the Cy-UCNPs. Because of the depletion of GSH, the UCL intensity within these cells all decreased obviously, and the degree of UCL decrease corresponded to the amount of NMM (Figure 4b− d). For the fourth group in which 1 mM exogenous GSH was introduced into the biothiol-depleted (by 5 mM NMM) HeLa cells, the intracellular UCL was recovered as expected (Figure 4e). HeLa cells were also incubated with PEG-UCNPs without loading Cy-GSH. The cells showed extremely weak UCL (Figure 4f), indicating that UCL enhancement is indeed caused by the reaction of Cy-GSH with GSH. Detection of GSH Level in Mice Liver. We further evaluated the capability of this UC nanoprobe for detection of GSH in more complicated biological environments, i.e., animal tissues. To this end, the mouse with acetaminophen (APAP)induced hepatotoxicity was chosen as the experimental model, considering the previously reported depletion of GSH in the impaired liver cells.50,51 As shown in Figure 5a, clear UCL signal was observed in the liver tissue of the mice i.p. injected with Cy-UCNPs, owing to the high concentration of GSH in normal mouse liver (this is similar to the HeLa cells experiments). For the two hepatotoxicity groups, mice were pretreated with different concentrations of APAP for 0.5 h before injection with the same amount of Cy-UCNPs. The liver showed a stepwise decrease of UCL intensity depending on the dosage of APAP (Figure 5b,c). According to another previous study, the decrease of GSH concentration in this liver damage model can be suppressed though pretreating the animal with α-lipoic acid (α-LA).52 Therefore, we designed two more groups of mice to study the protective effect of α-LA on the hepatotoxicity. For these two groups, the mice were pretreated with different concentrations of α-LA before the

probe after reaction with GSH kept stable over more than 20 h (Figure S13c). These results imply the possibility of using this UC nanoprobe for biological studies. To preclude other possible mechanisms of the UCL enhancement, we checked direct effect of GSH on the luminescence of PEG-UCNPs. GSH showed negligible influence on UCL with its concentration up to 5 mM (Figure S14a). Absorption spectra of Cy-GSH in the presence of GSH were also measured, so as to examine whether the UCL enhancement could result from the inhibition or reduction of the inner filter effect. Upon the reaction with GSH, the absorbance of Cy-GSH around 800 nm was enhanced (Figure S14b). This means the inner filter effect was further strengthened, which should lower the UCL intensity to some extent because of the further weakened excitation, but has no way to enhance the UCL. The energy transfer efficiency from Cy-GSH to Nd after reacting was studied. F-SG assembled with NaYF4:Yb,Er@NaYF4,Nd shows weaker fluorescence than that assembled with NaYF4@NaYF4 (Figure S15). The energy transfer efficiency is calculated as 34% according to the equation ET = 1 − FNd/FY, in which ET represents energy transfer efficiency, FNd and FY represent the fluorescence intensities of F-SG assembled with NaYF4:Yb,Er@NaYF4:Nd and NaYF4@NaYF4, respectively. Combining all the above experimental results, we can conclude that the UCL enhancement was an outcome of GSH-mediated sensitization by the dye. Monitoring GSH in Living Cells. Having confirmed the design strategy and optimized the operation conditions of this new UC nanoprobe, we then investigated its applicability in cellular imaging. To ensure the security of Cy-UCNPs, we first performed methyl thiazolyl tetrazolium (MTT) assay to check its cytotoxicity to living cells. The cell viability showed no obvious change after incubation for 12 h at 37 °C, even with the UC nanoprobe concentration up to 0.6 mg/mL (Figure S16), suggesting a quite low cytotoxicity of the UC nanoprobe. The single-cell Z-scanning experiment was conducted to study the uptake and distribution of the probe by living cells. Figure S17 shows that Cy-UCNPs were efficiently uptaken into cells and well distributed in the cytosol, probably arising from endocytosis facilitated by dyneins.47 To study the distribution of Cy-UCNPs in living cells, HeLa cells were costained with MitoTracker Red CMXRos (a commercial mitochondrial marker) and Lyso-Tracker Red (a commercial Lysosome 14700

DOI: 10.1021/jacs.8b07329 J. Am. Chem. Soc. 2018, 140, 14696−14703

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Journal of the American Chemical Society

Figure 6. In vivo UCL images after injection of 50 μL Cy-UCNPs (2 mg/100 g body weight) for 1 h in tumor tissues (red arrows) and opposite normal tissues (green arrows). The mice were preintravenously injected with 50 μL saline (a) or 50 μL NMM (2 mg/100 g body weight) (b) 20 min before Cy-UCNPs injection. (c) Normalized UCL intensities of tumor sites and normal sites of images a and b. The error bars are acquired with 3 mice each group.

UC nanoprobe employing a target-modulated dye-sensitization principle. Moreover, the Cy-UCNPs probe can readily be extended to develop other probes for different targets by simply changing the recognition domain of the dye. Our efforts may offer a useful alternative to the construction of UC nanoprobes and endow UCNPs-based tools with improved powers in biological and medical research in the future.

treatment with same amount of APAP and Cy-UCNPs. The UCL intensities of these two liver (Figure 5d,e), as excepted, were significantly enhanced compared to the group injected with the same dosage of APAP and the intensity was corresponding to the amount of α-LA injected (compare Figure 5d,e with 5c). In addition, the mouse injected with only physiological saline showed ignorable UCL signal (Figure 5f). Taken together, the above results suggest that Cy-UCNPs can be a promising probe for GSH detection in biological samples. In Vivo Imaging of GSH in Mice. Ultimately, we applied Cy-UCNPs to the in vivo detection of GSH in a tumor-bearing mouse. Cy-UCNPs were injected intratumorally, and applied subcutaneously on the opposite normal side of the same mouse as a control. As shown in Figure 6a, the intratumorally administered Cy-UCNPs accumulated in the tumors and displayed stronger UCL as we anticipated, due to the higher GSH levels in tumors than normal tissues. To further verify that the increased UCL signal was induced by GSH, another tumor-bearing mouse was treated with 2 mg/100 g body weight NMM for 20 min before Cy-UCNPs loading. As shown in Figure 6b, this mouse exhibited weaker UCL both in tumor and in normal tissue compared to the one only injected with Cy-UCNPs, because of the depletion of GSH by NMM (similar to the results of the above cell experiments). After in vivo imaging, the mice were dissected and the major organs including heart, liver, spleen, lung, kidney and also the tumor were harvested to study the distribution of Cy-UCNPs in the whole body. As seen in Figure S19a, the tumor showed the strongest UCL, indicating the probes were mostly accumulated in the tumor. The liver also exhibited obvious UCL emission because the nanoprobe can transfer to liver through metabolism,53 whereas the other organs did not show detectable UCL. And these results were identical with those by elemental analysis (Figure S19b).

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EXPERIMENTAL SECTION

Synthesis of Cy-GSH. The synthetic routine of Cy-GSH is shown in Figure S20. Compound 3. Compound 1 (4 g, 25.2 mmol) and compound 2 (9.6 g, 36.4 mmol) were dissolved in toluene and refluxed in N2 atmosphere overnight. After the reaction, the solvent was evaporated under reduced pressure. The crude compound was purified by column chromatography on silica gel to obtain 3 (9.9 g, 93% yield) as a brown oil. 1H NMR (400 MHz, d6-DMSO): δ 11.99 (s, 1H), 8.03−7.92 (m, 1H), 7.85 (dd, J = 5.7, 2.9 Hz, 1H), 7.70−7.55 (m, 2H), 4.49−4.38 (m, 2H), 2.83 (s, 3H), 2.18 (t, J = 7.3 Hz, 2H), 1.86−1.74 (m, 2H), 1.53 (s, 6H), 1.50−1.38 (m, 4H), 1.24 (s, 10H). Compound 5. Compound 5 was synthesized as reported.54 Compound 6. Compound 3 (560 mg, 1.32 mmol) and compound 5 (123 mg, 0.71 mmol) were dissolved in the mixture solvent of 1butanol/toluene (v:v = 10:1) and stirred at 120 °C for 12 h. Then, the solvent was removed under reduced pressure. The resulting residue was purified by silica gel column chromatography with gradient CH2Cl2 to CH2Cl2/MeOH eluent to give 6 (320 mg, 43% yield) as a dark green solid. 1H NMR (400 MHz, d6-DMSO): δ 8.26 (d, J = 14.2 Hz, 2H), 7.64 (d, J = 7.4 Hz, 2H), 7.45 (q, J = 8.0 Hz, 4H), 7.29 (t, J = 7.1 Hz, 2H), 6.33 (d, J = 14.2 Hz, 2H), 4.22 (s, 4H), 3.98 (t, J = 6.5 Hz, 4H), 2.70 (s, 4H), 2.25 (t, J = 7.3 Hz, 4H), 1.86 (s, 2H), 1.72 (d, J = 6.6 Hz, 4H), 1.67 (s, 12H), 1.50 (dd, J = 14.4, 6.6 Hz, 8H), 1.30 (dd, J = 15.0, 7.4 Hz, 28H), 0.85 (d, J = 7.4 Hz, 6H). Compound 9. 1 M HCl aqueous was added to compound 7 (1 g, 7.24 mmol) in water at 0 °C under stirring. NaNO2 aqueous was slowly added to the above mixture, and the solution was kept stirring for 1 h. This mixture was slowly added to the aqueous solution of NaOH and compound 8 (676.2 mg, 7.24 mmol) at 0 °C followed by stirring for another 1 h. After acidification by diluted HCl, the reaction system was filtered and washed with water and EtOAc to obtain 9 (714 mg, 41% yield) as an orange solid. 1H NMR (400 MHz, d6-DMSO): δ 10.65 (s, 1H), 8.41 (d, J = 8.4 Hz, 2H), 8.00 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.3 Hz, 2H), 6.99 (d, J = 8.3 Hz, 2H). Cy-GSH. NaH (2 mg, 0.05 mmol) was added to a solution of compound 9 (12 mg, 0.049 mmol) in anhydrous DMF with stirring for 0.5 h. Then, compound 6 (40 mg, 0.039 mmol) was added to the mixture and heated to 80 °C in N2 atmosphere. After reaction overnight, the mixture was precipitated by diethyl ether and filtered, followed by purification by silica gel column chromatography with CH2Cl2 to CH2Cl2/MeOH as eluent to give Cy-GSH (23 mg, 48%

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CONCLUSION In summary, we put forward a new strategy to construct UC nanoprobes using a target-modulated sensitizing switch. This design strategy gets rid of the dependence on the efficiency of upconversion luminescence quenching, thus breaking through the signal-to-background limit of UC nanoprobes. For the proof-of-concept GSH UC nanoprobe, a maximum SBR of 30fold was achieved, significantly larger than that of existing UC nanoprobes (normally