capable of palladium imaging at the cellular level with a series of dynamic changes. According to the results of cytotoxicity test
accomplished by SRB cytotoxicity assay, the hypoxia chemiluminescent probe is low-toxic and fine-biocompatible (Fig. S4 in
Supporting information).
Additionally, bioactivity of PCL towards palladium ion in commercial rabbit plasma (20 folds diluted with saline) was investigated.
For a 1-hour-incubation, as depicted in Fig. 6 and Fig. S5 (Supporting information), the chemiluminescence intensity augmented with
the concentrations of PdCl2 from 25 µmol/L to 100 µmol/L. These results indicated that PCL could detect palladium quantitatively
even in such complex biological samples. Unfortunately, 20-fold diluted rabbit plasma treated only with PCL released a
chemiluminescence signal over 2.5 times stronger than PCL treated with saline, which drove down the LOD of PCL. The disadvantage
may also be attributed to the instability of carbonate structure in complex biological samples. However, this study can still provide
some valuable data and experience for establishing a chemiluminescent system for the animal model.
Fig. 6. Palladium imaging in 20-fold diluted rabbit plasma using PCL. (A) Variation of chemiluminescence intensity in 60 min (100 μmol/L of PdCl2). (B)
Quantification of the chemiluminescent intensity of PCL for each condition.
Fig. 7. Palladium imaging in vivo using PCL. (A) Chemiluminescence imaging of PCL towards Pd2+ with various concentrations (0-100 μmol/L). (B)
Quantification of chemiluminescence intensity from the subcutaneous injection areas of the rude mice of triplicates (n = 4). (C) Variation of chemiluminescence
intensity within 35 min.
The capability of PCL for palladium(II) imaging in living animals was interrogated. We attempted to subcutaneous inject the 50 μL
of saline or palladium chloride (20 μmol/L, 50 μmol/L or 100 μmol/L) in saline. Following, 50 μL of PCL (1 mmol/L) in saline
containing 10% (v/v) Enhancer Emerald II was subcutaneous injected in situ respectively. Thereafter, subsequent chemiluminescence
intensity was recorded with living imaging system. As shown in Fig. 7, after injection of PCL, obviously enhancing emission signals
from injection regions in rude mice were observed, reaching peak values at about 30 min. The enhanced chemiluminescence intensity
lasted for about 5 min. As was expected, distinct enhancement of luminescence intensity can be observed visually by living imaging, as
injection amount of PdCl2 grew from 0 to 100 μmol/L.
In summary, herein we designed and prepared a palladium chemiluminescent probe (PCL) by using 1,2-dioxetane as the
chemiluminescence platform and butynyl moiety as the recognition unit. Exhibited desirable properties, including no requirement for
excitation light, low background noise, high sensitivity, high selectivity and the ability to function well in aqueous media, PCL is
capable of monitoring palladium ion visually in vitro, in cellulo and in vivo. Although the instability of carbonate structure in PCL may
drive down the LODs in the basic system or in complex biological samples, the exploration brought experience and expand the
palladium imaging toolkit and applications of chemiluminescence technology.
Acknowledgments
The present work was supported by grants from the Taishan Scholar Program at Shandong Province, the Qilu/Tang Scholar Program
at Shandong University, the Key Research and Development Project of Shandong Province (No. 2017CXGC1401) and the Major
Project of Science and Technology of Shandong Province (No. 2015ZDJS04001).
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