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Bioluminescent Probe for Detecting Mercury (II) in Living Mice Tianyu Jiang, Bowen Ke, Hui Chen, Weishan Wang, Lupei Du, Keqian Yang, and Minyong Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02200 • Publication Date (Web): 14 Jul 2016 Downloaded from http://pubs.acs.org on July 14, 2016
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Bioluminescent Probe for Detecting Mercury (II) in Living Mice Tianyu Jianga,‡, Bowen Keb,‡, Hui Chena,b, Weishan Wangc, Lupei Dua, Keqian Yangc and Minyong Lia, * a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, China. Tel./fax: +86-0531-8838-2076; E-mail:
[email protected] b
Laboratory of Anaesthesiology and Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China c
State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100190, China ABSTRACT: A novel bioluminescence probe for mercury (II) was obtained on the basis of the distinct deprotection reaction of the dithioacetal to decanal, so as to display suitable sensitivity and selectivity towards mercury (II) over other ions with bacterial bioluminescence signal. These experimental results indicated such a probe was a novel promising method for mercury (II) bioluminescence imaging in environmental and life sciences ex vivo and in vivo.
The development of chemical sensors for heavy metals attracted more and more attention because of their bioaccumulation, biomagnification, and persistence in the environment.1,2 The mercury ion (Hg2+), which usually exists in many industrial materials, is known as one of the most toxic metal ions both for human and environment. Recently, a lot of fluorescent and colorimetric probes for detecting mercury (II) have been designed and reported, and some of them have potential to be applied in biological and environment mercury (II) detection.1-11 Moreover, according to the organic chemistry, mercury (II) promotes the deprotection reaction of dithioacetal back to the previous aldehyde, which is considered to be a selective reaction towards mercury(II) and the basis of probes design strategy. Bioluminescent imaging (BLI) is a well-established optical technique and is widely applied in the detection and imaging of biological process in vitro and in vivo12-14. In general, bioluminescence is generated by the luciferasecatalyzed reaction of luciferin with or without co-factors. Compared with fluorescence, bioluminescence does not demand an excitation light source so as to have low background interference and high signal-to-noise ratios. Diverse probes for small molecules detection based on bioluminescence have been developed such as probes based on firefly luciferin for detecting fluoride ion15, hydrogen sulfide16, hydrogen peroxide12. To the best of our knowledge, BLI-based mercury(II) probe in imaging is still unavailable. To fill this gap, herein we report the first development of bioluminescent probe for mercury(II) detection, as well as its initial imaging application.
The Vibrio is one of the major genera of luminous bacteria, and its bioluminescence reaction starts with oxidation of reduced flavin and a long-chain aliphatic (C10-C12) aldehyde with the existence of the corresponding luciferase.17-19 It is considerable that the Vibrio bacterial luciferase is encoded by lux gene cassette that consists of a set of genes, in which the aldehyde decanal is regarded as a luciferin 20-22. Moreover, either full or parts of them (luxAB) could express in alternative host organisms such as Escherichia coli20,22,23, which could become a useful tool in BLI. When decanal comes to design of reaction-based mercury (II) probe, the specific deprotection of dithioacetal by mercury (II) has been employed. Ethanethiol converts decanal to a caged luciferin via the protection reaction. When a caged luciferin is involved in a bacterial bioluminescent system, it will not trigger bioluminescence. Moreover, only upon the addition of mercury (II) that promotes the deprotection reaction of the dithioacetal, the free decanal will be released to provoke the detectable bacterial bioluminescence signal. To demonstrate this hypothesis, we designed and synthesized one probe by utilizing protection reaction between ethanethiol and decanal. It should be noted that this probe can be conveniently prepared after reacting of decanal with ethanedithiol and boron trifluoride etherate (see Scheme S1 in supporting information). The structural characterization information and the preparation of the E. coli culture expressing bacterial luciferase goes into details in the supporting information.
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Scheme 1: The mechanism of the bioluminescent probe for detection of Hg2+.
N o r m a liz e d B L in te n s ity
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Figure 2: the liner bioluminescence of the probe with 2.5-15 μM of Hg2+. When such a bioluminescent probe in hands, its efficiency to detect mercury(II) was firstly evaluated in an aqueous buffer system (50 mM Tris-HCl, pH 7.4). The differences of the bioluminescence intensities achieved by the released decanal reacting with E. coli culture expressing bacterial luciferase can reveal the efficiency and selectivity of the probe. The results (Figure 1) met our expectation that the probe exhibited a significant increase in bioluminescence intensity with an increase of mercury(II) concertation after just a few minutes’ incubation. However, this phenomenon occurred when mercury (II) was not over the concentration of 15 μM. And the bioluminescence intensity would not increase when the concentration of mercury (II) was over 15 μM that could be caused by the intrinsic toxicity of mercury (II). Moreover, a linear regression equation for mercury(II), B = 2.486x - 6.169 (R2 = 0.9925), was obtained when the concentration of mercury(II) at the range of 2.5-15 μM (Figure 2), where B refers to the ratio of bioluminescence intensity and x refers to the concertation of mercury (II). Overall, these data indicated that our probe has fast and distinct response ability of mercury(II).
Figure 1: (A) The bioluminescence imaging of the probe in incubation with 0-15 μM of Hg2+. (B) The quantification of the total flux (photon/s) of the probe with various concentrations of Hg2+. The final concentration of the probe is 50 μM.
It is essential for a probe with potential application to have a highly selective response to the target species over other potentially competing species. Hence, we investigated the selectivity of our probe towards mercury (II) over other cations. As expected and shown in Figure 3, there was no remarkable bioluminescence signal in the treatment of the common cations such as Fe2+, Fe3+, Ca2+, Mn2+, Ba2+, Cd2+, Mg2+, Zn2+, Cu2+, Ag+, Co2+ and Al3+. In comparison, mercury(II) can induce our probe to generate a ca. 7-fold significant bioluminescence signal enhancement.
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Figure 3. The bioluminescence imaging of cations selectivity of the probe (50 μM) towards various cations (Fe2+, Fe3+, Ca2+, Mn2+, Ba2+, Cd2+, Mg2+, Zn2+, Cu2+, Ag+, Co2+, Al3+ and Hg2+) at 20 μM or the probe only. The bioluminescence spectra of our probe treated with different cations were also measured. As depicted in Figure 4, the bioluminescence emission was detected only when the bacterial luciferase reacted with free decanal that is released from incubation of our probe with mercury(II). And the bioluminescence spectrum of the probe in the treatment of mercury (II) is consistent with the bioluminescence spectrum of decanal. There was no obvious bioluminescence emission occurring when the probe in the treatment of other cations. In addition, the probe’s ability to detect mercury(II) was evaluated by using HPLC. The comparison of the probe alone, the probe in the treatment of mercury(II), the decanal alone and the mix of the probe and decanal were displayed in Figure S1. These results also confirmed that our probe could convert back to decanal with the addition of mercury (II). Both the results of imaging and spectra measurement revealed that our probe is a highly selective bioluminescence probe for mercury (II).
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Figure 4: The normalized bioluminescence spectra of (A) the probe in the treatment of Hg2+ and decanal alone respectively, (B) the probe in the treatment of Hg2+ and other cations. The concentration of each ion is 25μM, the concentration of the probe and decanal is respective 50μM. Further, we examined the mercury(II) by use of the probe in mouse blood serum. The nude mice were treated with HgCl2 at 10 mg/kg/day, 20 mg/kg/day and normal saline, respectively. After collection of blood serum, the serum samples were treated with the probe and bacterial luciferase sequentially. As we can see in Figure 5, the bioluminescence increased moderately with the increase of mercury(II). Though the whole total flux was low overall, the results also indicates that our probe has the potential to monitor the mercury(II) in blood serum to some extent.
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Figure 5: The bioluminescence of mercury(II) tested by the probe in mouse blood serum. The mice were injected with HgCl2 at 10 mg/kg/day, 20 mg/kg/day and the same volume of normal saline, respectively. Considering the respectable performance of our probe in vitro experiments, we decided to apply our probe in in vivo imaging of mercury (II) by using nude mice model. After subcutaneously injected with solution of mercury (II) chloride, the mice were subcutaneously injected with the probe in situ quickly and then were subcutaneously injected with E. coli culture expressing the bacterial luciferase in situ quickly. As shown in Figure 6, the bioluminescence signal triggered by mercury (II) can be detected in whole animal imaging. And the bioluminescence intensity was different with the difference of the mercury (II) concentration. It is evident that little bioluminescence was observed in nude mice only subcutaneously injected by the probe without injection of mercury (II) chloride. The interesting results suggest that our probe has the ability to visualize the level of mercury(II) in living animals.
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mice. All assays were performed in triplicate and represented as the mean ± SD **P < 0.05. In conclusion, herein we report the design, synthesis and preliminary biological application of the first bioluminescence probe for the detection of mercury (II). On the basis of deprotection of dithioacetal by mercury(II), the probe exhibited a sensitive and selective “turn-on” response to mercury (II) over other ions. The probe triggered the linear bioluminescence signal in the response of the mercury (II)–dependent concentrations within certain ranges. And this probe can be successfully applied in the whole animal imaging. In addition, the application of bacterial bioluminescence system that we choose into the bioluminescence probes is rare. However, we made it into realization with the simple synthesis of the probe from decanal. All in all, this bioluminescent probe is believed to be a potential tool to detect mercury (II) in the biological and environmental application as well as providing a guideline to develop the novel bioluminescent method that can explore deeply the complicated contribution of mercury (II) to physiological and pathological processes.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The synthesis of probe, experimental procedure, HPLC spectrum, NMR spectra and HR-MS spectrum are supplied as Supporting Information. (PDF)
AUTHOR INFORMATION Corresponding Author * Tel./fax: +86-531-8838-2076; e-mail:
[email protected] ‡These authors contributed equally.
Notes The authors declare no competing financial interest.
ACKNOWLEDGMENT This work was supported by grants from the National Program on Key Basic Research Project (no. 2013CB734000), the Qilu Professorship in Shandong University, the National Natural Science Foundation of China (no. 81370085), the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT13028), the Major Project of Science and Technology of Shandong Province (No. 2015ZDJS04001), the Shandong Key Research & Development Project (No. 2015GSF118166), and the Independent Innovation Foundation of Shandong University, IIFSDU (no. 2014JC008).
Figure 6: (A) The bioluminescence imaging of the probe with normal saline or various concentrations of Hg2+ in living nude mice. (B) The quantification of the total flux (photon/s) of the probe with normal saline or various concentrations of Hg2+ from the bright area of the nude
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