Catalytic Chemiluminescence Polymer Dots for Ultrasensitive in vivo

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Catalytic Chemiluminescence Polymer Dots for Ultrasensitive in vivo Imaging of Intrinsic Reactive Oxygen Species in Mice Lvping Cai, Liyun Deng, Xiangyi Huang, and Jicun Ren Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01188 • Publication Date (Web): 07 May 2018 Downloaded from http://pubs.acs.org on May 7, 2018

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Analytical Chemistry

Catalytic Chemiluminescence Polymer Dots for Ultrasensitive in vivo Imaging of Intrinsic Reactive Oxygen Species in Mice Lvping Cai, Liyun Deng, Xiangyi Huang*, and Jicun Ren* School of Chemistry and Chemical Engineering, State Key Laboratory of Metal Matrix Composites, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, People’s Republic of China ABSTRACT: Chemiluminescence (CL) is a promising bioimaging method due to no interferences of light source and autofluorescence. However, compared to fluorescent emission, most CL reactions show short emission time and wavelength and weak emission intensity, which limit their applications in in vivo imaging. Here, we report mimic-enzyme catalytic CL polymer dots (heminPdots) consisting of hemin and fluorescent conjugated polymer based on chemiluminescence resonance energy transfer. HeminPdots show about 700-times enhanced CL and over10 hour light emission in the presence of CL substrates and H2O2. These properties are mainly due to high-catalytic activity of hemin-Pdots and slow-diffusion-controlled heterogeneous reaction. Hemin-Pdots also possess excellent biocompatibility, good stability, emission wavelength redshift and ultrasensitive response to reactive oxygen species (ROS), and they were successfully used for real-time imaging ROS levels in the peritoneal cavity and normal and tumor tissues of mice. Hemin-Pdots as new CL probes have wide-applications in bioassays, bioimaging, and photodynamic therapy.

Chemiluminescence (CL) imaging is a promising method for the noninvasive detection of important biomolecules in cells and animals, and it provides spatial and temporal information of biological processes.1−3 However, there are only a few reports about in vivo CL imaging4−18 compared with the widelyused fluorescence (FL) imaging. The main reasons are most CL reactions suffering from weak CL intensity, short emission duration and wavelength, which limit their applications in in vivo imaging. Recently, chemiluminescence resonance energy transfer (CRET) and nanomaterials are used to redshift the CL wavelength. We for the first time proposed CRET to shift the CL wavelength (425 nm) to near infrared by using quantum dots as energy acceptors.19 Up to now, some nanomaterials have been used as CRET accepters (e.g., quantum dots, conjugated polymer-based polymer dots (Pdots) and so on) to significantly improve the energy transfer efficiency.19−32 Among these energy acceptors, Pdots are attractive candidates for their distinguished signal amplification along the backbone, excellent chemical stability, and adjustable optical performance. CRET coupled with conjugated polymer dots have been used for in vivo imaging by embedding or covalently linking CL substrates in Pdots,27−31 or by conjugating CL catalysts such as horseradish peroxidase (HRP) onto the surface of Pdots.32 Although the CL method has made great progress, it is still a huge challenging to develop new CL probes with strong CL intensity and long emission duration and wavelength in CL bioimaging. Compared to direct CL mode, catalytic CL mode has ultrahigh sensitivity and long CL duration by labeling of a CL catalyst (such as HRP) to a target due to the signal amplification of enzyme catalysis, and it is widely used for in clinical diagnosis such as immunoassay. The luminol/hydrogen peroxide CL reaction catalyzed by HRP is one of the most sensitive CL reactions. In capillary electrophoresis with this CL detection,

the detection limit of HRP was below 10−19 mol.33 However, till now, there are few reports for in vivo CL imaging via catalytic CL mode and they are usually used for detecting the intrinsic CL enzyme activity.5,18,24 Wang et al. have conjugated the CL catalyst HRP onto the surface of the Pdots for improving CL properties and on-site imaging of cancer cells.32 Recently, Cui et al.34 reported firefly-mimicking intensive and long lasting CL hydrogels using Co2+ ions as CL reaction catalysts, and this CL hydrogels showed over 150 hour emission time. The excellent CL property of hydrogels was mainly attributed to slow-diffusion-controlled heterogeneous reaction dynamics. These results have documented that catalytic CL mode has great potential to enhance CL intensity and prolong emission time. Reactive oxygen species (ROS) widely exist in living organisms and play active roles in various physiological processes including cell growth, immune response and senescence.35,36 Excessive ROS are often implicated as potent inducers of oxidative damage as well as mediators of ageing, inflammation and cancers.37 Consequently, in vivo imaging of intrinsic ROS has aroused researchers’ great interest because it can provide intuitive spatial and temporal information.27−30,38−41 CL imaging is a promising method for the noninvasive detection of important biomolecules in vivo for its high sensitivity and no autofluorescence. However, previously reported direct CL mode, have produced low signal-to-noise ratio in identical LPS-treated mice model and usually could not provide in situ visualizing ROS level differences between normal and tumor tissues of mice Corresponding Author

* Phone: +86-21-54746001. Fax: +86-21-54741297. Email: [email protected]

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due to their low CL intensity.27 The catalytic CL mode may be a competitive method for in vivo imaging ultralow level of biomolecules because of its ultra-high sensitivity and long CL duration. Unfortunately, to the best of our knowledge, there is no report about in vivo CL imaging of ROS by using catalytic CL nanoprobe. Here, we first present ultra-intensive and long-time catalytic CL polymer dots (hemin-Pdots) using hemin as CL catalysts and fluorescent polymer as energy acceptors. Hemin is a HRPmimic and possesses a high catalytic activity in CL reaction due to its structure of iron (III) protoporphyrin IX similar to HRP. More importantly, hemin is a highly hydrophobic compound unlike HRP, and it can be easily embedded into the Pdots. The catalytic CL principle of hemin-Pdots is based on the hemin-catalyzed reaction of CL substrates and H2O2 and CRET effects. In this CRET system, CL substrates in solution are used as energy donors and fluorescent conjugated polymers are used as energy acceptors. The hemin-Pdots show about 700-times enhanced CL signal, and the signal to noise ratio is over 1,300 after 10 hour emission. Hemin-Pdots also possess excellent biocompatibility, good stability and ultrasensitive response to ROS, and are successfully used for real-time imaging ROS levels in the peritoneal cavity and normal and tumor tissues of mice.

EXPERIMENTAL SECTION Synthesis of Hemin-Pdots. The hemin-Pdots were synthesized by using nano-precipitation.42,43 PS-g-PEG-COOH (0.25 mg), hemin (5 µg), and PFPV (0.375 mg) were dissolved into a THF (5.0 mL) solution. The mixture was then rapidly injected into 10 mL water under continuous sonication with a sonicator bath for 2 min. After THF was removed by partial vacuum evaporation, the aqueous solution was filtered through a 0.22 µm cellulose membrane filter. The formed hemin-Pdot suspension was finally concentrated to different concentrations by ultrafiltration and kept at 4 °C for the following experiments. The concentration of hemin-Pdots was measured by fluorescence correlation spectroscopy (FCS). The doping concentration of hemin was estimated to be 20∼30 hemin molecules per nanoparticle. In vitro Imaging of Hemin-Pdots. For CL measurement, 100 µL hemin-Pdots (50 µg mL−1) and L012 ((8-amino-5chloro-7-phenylpyrido [3,4-d] pyridazine-1,4 (2H,3H) dione), 0.25 mM) in PBS (10 mM, pH 7.4) was placed in a black 96well plate. After addition of 100 µL H2O2 (0, 0.5, 1, 2, 4, 6, 8, and 10 µM), the CL was continuously acquired using an IVIS Spectrum II preclinical in vivo imaging system with open filter and the exposure time was 1 minute. The intensities were calculated from a region of interest and plotted as a function of time. We investigated the CL responses of hemin-Pdots in human emborynic kidney cell (HEK 293) and human glioblastoma cell line (U87MG) extracts. One hundred microliter HEK 293 or U87MG extracts was placed in a black 96-well plate, respectively. After addition of 100 µL hemin-Pdots (50 µg mL−1) in PBS (10 mM, pH 7.4), the CL was continuously acquired using an IVIS Spectrum II preclinical in vivo imaging system with open filter and the exposure time was 1 minute. Animal Models of Drug-Induced Inflammation and in vivo Imaging. Animal procedures were approved by the Ethical Committee of Shanghai Jiao Tong University. In LPSinduced inflammation model, six groups of mice were treated

with different conditions. In vivo imaging of endogenous ROS in the mouse model of LPS-induced inflammation used L012 or L012 and hemin-Pdots as imaging agents. (I) PBS (300 µL) was injected into abdomen, followed by injection of L012 (0.25 mM in 300 µL PBS) 4 h later. (II) LPS (2 mg mL−1 in 300 µL PBS) was injected into abdomen, followed by injection of L012 (0.25 mM in 300 µL PBS) 4 h later. (III) LPS (2 mg mL−1 in 300 µL PBS) was injected into abdomen, followed by injection GSH (10 mg mL−1 in 300 µL PBS) 3 h later. 1 h later, L012 (0.25 mM in 300 µL PBS) was injected. (IV) PBS (300 µL) was injected into abdomen, followed by injection of L012 (0.25 mM) and Pdots (50 µg mL−1) in 300 µL PBS 4 h later. (V) LPS (2 mg mL−1, in 300 µL PBS) was injected into abdomen, followed by injection of L012 (0.25 mM) and Pdots (50 µg mL−1) in 300 µL PBS 4 h later. (VI) LPS (2 mg mL−1 in 300 µL PBS) was injected into abdomen, followed by injection GSH (10 mg mL−1 in 300 µL PBS) 3 h later. 1 h later, L012 (0.25 mM) and Pdots (50 µg mL−1 in 300 µL PBS) was injected. (n = 3) Then, mice were anesthetized with 2% isoflurane. CL images were acquired by an IVIS Lumina II in vivo imaging system with open filter. Tumor Implantation and in vivo Imaging. Animal procedures were approved by the Ethical Committee of Shanghai Jiao Tong University. Tumor cells were harvested by incubation with 0.05% trypsin-EDTA when they reached near confluence. Cells were pelleted by centrifugation and resuspended in sterile PBS. U87MG cells (2 × 106 cells/site) were implanted subcutaneously into the left shoulder of four- to five-weekold female nude mice (Chinese Academy of Sciences of Shanghai). When the tumors reached the size of 4 to 8 mm in diameter (two to three weeks after implantation), the tumorbearing mice were subjected to imaging studies. For in vivo chemiluminescence imaging, the mice were imaged after subcutaneous injection of L012 and hemin-Pdots (0.25 mM for L012 and 50 µg mL−1 for hemin-Pdots in 50 µL of PBS). Images were acquired without filters.

RESULTS AND DISCUSSION The Synthesis and Characterization of Hemin-Pdots. Scheme 1 shows the strategy for preparation of hemin-Pdots and their catalytic CL principle. Hemin-Pdots were prepared in aqueous medium using nanoprecipitation of conjugated polymer PFPV, amphiphilic block copolymer PS-PEG-COOH and hemin, and the procedure was described in the experimental section. In the coprecipitation, hemin was embedded by conjugated polymer chains to form compact spherical nanoparticles due to its high hydrophobicity. The scheme of hemin-Pdots catalyzed L012-H2O2 CL reaction is shown in Figure S1. L012 is a kind of derivatives of luminol and it exhibits a 100-fold higher CL intensity than luminol in physiological condition.12,25 Therefore, we choose L012 for in vivo imaging in this work. Firstly, we systematically optimized the synthesis conditions of hemin-Pdots. The ratios of PFPV to PS-PEGCOOH have been optimized. We found that the quantum yield of hemin-Pdots is highest when the ratio of PFPV to PS-PEGCOOH was 1.5 (shown in Figure S2). CL signal increased with an increase in the concentration of hemin in the synthesis reaction solution, but the CRET ratio (the ratio of the acceptor emission to the donor emission) decreased with the concentration of hemin in the synthesis reaction solution because of the FL quenching effect of


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Analytical Chemistry

Scheme 1. A) Schematic illustration for preparation of hemin-Pdots and their principle of the catalytic CL mode, B) CL images of L012 and H2O2 catalyzed by hemin-Pdots. The CL image of blank is from the CL reaction of L012 and H2O2 without hemin-Pdots (1 min).

Figure 1. A) TEM image of hemin-Pdots. The scale bar was 100 nm. B) The FCS auto-correlation curve of hemin-Pdots (upper) and its residual curve (under). C) Absorption spectrum (black line) and emission spectrum (red line) of hemin-Pdots. D) Absorption spectrum (black line) of hemin-Pdots, and CL spectra of L012 (red line)and hemin-Pdots (blue line). The intensity of absorption and CL spectra are − normalized. CL is detected in the presence of 1 mM H2O2 and 0.25 mM L012 without (red line) or with (blue line) 25 µg mL 1 heminPdots. Peak a is from L012 and Peak b is from hemin-Pdots.



hemin to PEPV (shown in Figure S3 and Figure S4). Therefore, we chose 0.5 µg mL−1 hemin in the synthesis reaction solution for the experiment in order to obtain both relatively strong CL intensity and high CRET ratio of hemin-Pdots. And then, we characterized the morphology and optical properties of hemin-Pdots by using the transmission electron microscopy (TEM), dynamic light scattering (DLS), FCS, UV–Vis absorption, FL and CL spectroscopies. As is shown in Figure 1A, Pdots are spherical morphology with average diameters about 22 ± 3 nm. The TEM result is a little smaller than the results obtained in FCS and DLS experiments (30 ± 4 nm, Figure 1B and Figure S5) since these methods provided the hydrodynamic diameters of the hemin-Pdots. The potentiometric analysis displayed that zeta potential of Pdots were −19.0 ± 0.5 mV, and their negative charge was mainly due to hydrophilic carboxylic acid groups on the surface of heminPdots. FCS measurements documented that the concentration of hemin-Pdots in the synthesis reaction solution was about 10−20 nM and the doping amount of hemin was 20−30 hemin molecules per nanoparticle. The UV–Vis absorption spectra of Pdots (PEPV polymer) and hemin-Pdots indicated the broad absorption of PFPV (300–530 nm) and a peak at 455 nm (Figure 1C and Figure S6). Importantly, the CL spectrum of L012 (λmax, 470 nm) overlaps well with the absorption spectrum of PFPV (Figure 1C), which indicates that hemin-Pdots probably possess high CRET efficiency. In the presence of hydrogen peroxide, intense luminescence of hemin-Pdots was observed both at 470 nm and longer wavelength 540 nm (Figure 1D), confirming efficient CRET between L012 and PFPV. The CRET ratio is about 1.5, and the data manifest that the prepared hemin-Pdots possess high CRET efficiency. The possibility of hemin leakage from the hemin-Pdots has been studied by comparison of the CL signal before and after ultra-filtration of hemin-Pdot solutions. As shown in Figure S7, the leakage of hemin from the hemin-Pdots can be ignored since the change of the CL signal is very small after ultra-filtration.

Figure 2. A) CL images of hemin-Pdots (the left), L012 (the middle), and CPPO-Pdots (the right). B) The integrated CL intensity of hemin-Pdots, L012, and CPPO-Pdots. CL is detected in the presence of 1 mM H2O2 and 0.25 mM L012 with or without 25 µg − mL 1 hemin-Pdots. The concentration of CPPO-Pdots is 50 µg − mL 1. The reaction buffer is PBS (pH 7.4).

The CL Properties, Stability and Biocompatibility of Hemin-Pdots. We investigated systematically the catalytic activity, stability and biocompatibility of hemin-Pdots. The concentration of L012 has been optimized and 0.25 mM L012

was chose for following experiments because of high CL intensity (shown in Figure S8). As shown in Figure 2, the hemin-Pdots owned high peroxidase activity under physiological conditions, and their catalytic CL intensity was over 700 times stronger than direct CL modes of the L012 and the CPPOPdots (shown in Figure S9). The ultra-intensive CL is mainly due to a large number of catalysts in hemin-Pdots. We investigated the CL kinetics of direct CL mode and the hemin-Pdots catalytic CL mode, respectively. As depicted in Figure 3, CL intensities of L012 attenuated dramatically within 36 min (Figure 3C). However, the CL duration of hemin-Pdots obviously lasted over 10 hours, and the signal to noise ratio was still over 1,300 after 10 hour emission.

Figure 3. A) The CL images of L012. B) The CL images of hemin-Pdots catalyzed L012. C) The integrated CL intensity of L012. D) The integrated CL intensity of hemin-Pdots catalyzed L012. CL is detected in the presence of 10 mM H2O2 and 0.25 − mM L012 with or without 25 µg mL 1 hemin-Pdots. The reaction buffer is PBS (10 mM, pH 7.4).

Our above results demonstrate that hemin-Pdots show 700times enhancement CL and over 10 hour light emission in the presence of CL substrates and H2O2. Such ultra-intensive and long-lasting emission of the hemicn-Pdots is mainly attributed to high-catalytic activity of hemin-Pdots and slow-diffusioncontrolled heterogeneous reaction dynamics. In this study, hemin is used as a catalyst of CL reaction. Hemin is a HRPmimic, contains an iron (III) protoporphyrin IX, and its structure is similar to HRP. However, the free hemin molecules have a strong tendency to self-aggregate in an aqueous solution, which resulted in significant decrease in their catalytic activity.44 In order to resolve this problem, a variety of catalytic species such as peroxidase mimics DNAzymes have been prepared by binding of DNA aptamers to hemin.22 The DNAzymes show two orders of magnitude of peroxidase activity over the intrinsic catalytic capability of the hemin.22,45 The high activity of DNAzyme is mainly due to DNA aptamers offering a hydrophobic hemin-binding site and in this case the hemin dimerization and oligomerization equilibria are towards the active monomeric form. The situation of hemin in the Pdots is similar to DNAzyme and HRP since hemin molecules also have a hydrophobic environment and are welldistributed in the Pdtos. More importantly, there are about 20−30 hemin molecules in a single Pdot, and each hemin lo-


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Analytical Chemistry cated in the Pdot is an active site of CL reaction. When CL substrates diffuse to the active site, hemin catalyzed CL reaction generate strong CL emission.34,45 In addition, hemin-Pdots are nanoparticles different from enzyme molecules, and the hemin-Pdots catalyzed CL reaction belongs to heterogeneous catalysis system. Due to the slow diffusion of hemin-Pdots in solution and the slow diffusion of CL substrates (L012 and H2O2) in the nano-sized pores of Pdots, these slow-diffusioncontrolled processes resulted in the long-lasting CL. It was also reported that the attachment of catalyst metallocene to a rigid polymer resulted in an increase in the catalytic activity and the stability of catalyst.46 This is because the active site on the polymer was isolated and the catalyst poisoning was slowed down.46 Thus, in this case, hemin embedded in the Pdots exhibited unique heterogeneous catalytic activity on the CL reaction, leading to intensive and long-lasting emission. These attractive merits above are favorable for a practical application of in vivo biological imaging.

Figure 4. A) Specificity of chemiluminescence signal of heminPdots determined in the presence of various reactive species (1.0 mM). B) CL images of hemin-Pdots in response to indicated concentrations of H2O2 by an IVIS Lumina II in vivo imaging system with open filter. The exposure time is 1 min. C) Linear relationship between CL intensities of hemin-Pdots and concentrations of − H2O2 (0.5−10 µM). Hemin-Pdot is 25 µg mL 1 and L012 is 0.25 mM in 10 mM PBS buffer.

The storage experiments showed that hemin-Pdots kept strong catalytic activity within 30 days (shown in Figure S10). This remarkable stability of hemin-Pdots is mainly owed to the fact that hemin is a kind of stable chemical compounds unlike proteins such as enzymes. Futhermore, the fluorescence intensity of the conjugated polymer in the hemin-Pdots was almost unchanged when 0 to 50 mM H2O2 were added (shown in Figure S11). The cytotoxicity of the hemin-Pdots was evaluated by the MTT assay of U87MG and mouse macrophages mouse peritoneal macrophage cell line (RAW264.7), and no significant differences in cell viability were observed in the absence or presence of 5–100 µg mL−1 the hemin-Pdots (shown in Figure S12 and Figure S13). Furthermore, our experiment results demonstrated that hemin-Pdots were also nontoxic to mice up to 100 µg mL−1 (Figure S14). As shown in Figure 4A, heminPdots show the sensitive CL response to certain ROS such as H2O2, TBHP and ClO−, and these results suggest that heminPdots can be used to determine these ROS. Figure 4B and Figure 4C display the linear relation of CL intensities and the concentrations of H2O2 in the range from 0.5 to 10 µM, and the detection limit is 10 nM. These results indicated that hemin-Pdots was used for quantitative determination of H2O2 level.

In vitro and in vivo Imaging the ROS Levels by HeminPdots. The hemin-Pdots were firstly used to monitor intrinsic ROS level in HEK 293 (normal cells) and U87MG (tumor cells), respectively. As shown in Figure S15, CL intensity of the U87MG extracts was 1.7 times higher than that of HEK 293 extracts, reflecting higher level ROS in tumor cells. These in vitro data clearly demonstrate that hemin-Pdots have the capability of specifically sensing native ROS in biological samples and also differentiating normal and tumor cells by variant ROS levels. Furthermore, we investigated the ability of hemin-Pdot catalytic CL mode to image endogenously produced ROS in mice. As reported, excessively produced ROS is implicated in the development of numerous inflammatory diseases. Therefore, there is a great interest in imaging ROS in vivo because of its potential to act as a diagnostic method for inflammatory diseases.

Figure 5. A) In vivo imaging of endogenous ROS in the mouse model of LPS-induced inflammation with L012 (top, imaged 7 min after L012 administration) or L012 and hemin-Pdots (bottom, imaged 7 min after L012 and hemin-Pdots administration). (I) PBS was injected into abdomen, followed by injection of L012 4h later. (II) LPS was injected into abdomen, followed by injection of L012 4h later. (III) LPS was injected into abdomen, followed by injection GSH 3 h later. 1 h later, L012 was injected. (IV) PBS was injected into abdomen, followed by injection of L012 and hemin-Pdots 4h later. (V) LPS was injected into abdomen, followed by injection of L012 and Pdots 4h later. (VI) LPS was injected into abdomen, followed by injection GSH 3 h later. 1 h later, L012 and Pdots was injected. B) Quantification of CL signals calculated from the in vivo images in A. C) Quantitative CL intensities of the mouse model of LPS-induced inflammation with L012 or L012 and Pdots over time (n = 3). Black arrow indicates the respective time points shown in A.

Here, hemin-Pdot catalytic CL mode was used to evaluate the endogenous ROS in mice (as shown in Figure 5 and Figure S16,), generated by activated macrophages and neutrophils,47 in a lipopolysaccharide (LPS) model of acute inflammation.48 Different groups of mice were treated with intraperitoneally (i.p.) administrated saline (I), LPS (II), and LPS + GSH (III), respectively. Then, hemin-Pdots and L012 were injected (i.p.), and CL images were acquired. Notably, CL intensity in the LPS-treated group was (18 ± 5) × higher than that of only saline-treated group by using hemin-Pdot catalytic CL mode (Figure 5B). In contrast, the CL intensity of this catalytic CL mode was almost 10 times higher than that of



direct L012 CL mode. In addition, previously reported direct CL mode, has produced only 2-fold signal-to-noise ratio in identical LPS-treated mice model.27 Then, upon the injection of GSH to the LPS-pretreated mouse, the CL intensity weakened distinctly. Moreover, Figure 5C and Figure S16 demonstrate that hemin-Pdots have long luminescence duration, and are beneficial to continuously monitor ROS. To assess the practicability of hemin-Pdots to spy on intrinsic ROS in tumor tissues of mice, we constructed a human glioblastoma model of mice by injecting U87MG cells in forelimb armpit of mice. After 2-3weeks, a tumor mass of about 4 to 8 mm in diameter was obtained. In the CL imaging, L012 and hemin-Pdots were injected in tumor and normal tissues of the mice, respectively. Subsequently, we found that the CL intensity in tumor tissue was (7.9 ± 1.3) × higher than that of the normal tissue in hemin-Pdot catalytic CL mode, indicating noticeably higher levels of ROS in the tumor tissue (as shown in Figure 6 and Figure S17). However, we could not distinguish the tumor tissue from the normal tissue by direct L012 CL mode (as shown in Figure 6). Apparently, CL imaging quality and sensitivity were significantly upgraded owing to the effective CRET, high peroxidase activity of hemin-Pdots and without biological autofluorescence. These results above documented that hemin-Pdot catalytic CL mode has the great potential for imaging ROS-associated inflammatory diseases and cancers.

min-Pdots as labeling probes have great potential applications in biosensors, bioimaging, clinical diagnosis, and photodynamic therapy.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Experimental details for chemicals and measurements, the synthesis of CPPO-Pdots, investigation of the stabilities of heminPdots and CPPO-Pdots, optical responses of hemin-Pdots toward reactive species, cell culture, MTT Assay and in vivo toxicity experiments, as well as supplementary figures, and references (PDF).

Author Contributions All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21327004, 21675111) and the Natural Science Foundation of Shanghai (14ZR1423400).

REFERENCES

Figure 6. A) Representative images (pseudocolor) of mice in vivo normal tissue (I) and tumor tissue (II) followed by L012; Images of mice in vivo normal tissue (III) and tumor tissue (IV) followed by L012 and hemin-Pdots. The exposure time was 180 s after L012 or L012 and hemin-Pdots administration. B) Quantitative CL intensities of normal and tumor tissues administrated by L012 and hemin-Pdots over time (n = 3). Black arrow indicates the respective time points shown in A.

CONCLUSIONS In conclusion, we present catalytic CL hemin-Pdots with ultraintensive and long emission duration. The CL principle of hemin-Pdots is based on CRET between the catalytic CL of hemin and Pdots in the presence of ROS and CL substrates. The CL intensity of this catalytic mode is seven-hundred times more than the direct mode and the signal to noise ratio is still over 1,300 after 10 hour emission. The strong emission intensity, long emission duration, and wavelength are attributed to the high catalytic activity, high CRET efficiency and distinguished signal amplification of hemin-Pdots. The hemin-Pdots were successfully applied to visualize ROS levels in normal/inflammation tissues and differentiate native concentration of ROS between normal and tumor tissues of mice. Hemin-Pdots own excellent stability and biocompatibility, and they are also easy of synthesis and no need of modification and purification procedures. We believe the catalytic CL he-

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Catalytic Chemiluminescence Polymer Dots for Ultrasensitive in vivo

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