As a reliable,sensitive,convenient and non-invasive in vivo imaging technology,bioluminescence imaging (BLI) has been extensively applicable for detecting physiological and pathological processes in life sciences ^[1]. BLI have been widely used in monitoring cells and biomolecular processes in cellulo or in vivo, including pathogen detection,tumor growth and responses to therapy patterns of gene regulation,measurements of protein- protein interactions and ADMET (absorption,distribution,metabolism, excretion and toxicity) ^[2]. The longitudinal and duplicate imaging without the sacrifice of animal models is what BLI can achieve,and as a result,the processes of the living animals can be detected in real time and noninvasively other than an endpoint determination by killing the animals. Similarly,fluorescence imaging (FLI) have been well introduced and immensely used in detecting diversified living processes due to its low cost and high speed ^{[3, 4]}. Compared with FLI,without excitation light source,BLI also can avoid the effect of external lights in the absolute darkness. Since most of the cell- or animal-based models do not express luciferase,the interference of background signal does not exist. What is more,the sensitivity of BLI detection is determined by the emission spectra of bioluminescent reporters as well as the interaction with mammalian tissue ^[8].

However,there are some disadvantages in the study of bioluminescent imaging. The key limitation to developing bioluminogenic probes is the shortage of design strategy ^[5]. So far,almost all available bioluminogenic probes are designed as the ‘‘caged luciferin’’,in which the 60-hydroxyl (or 60-amino) or 4- carboxyl position of luciferin (or aminoluciferin) was caged by some specific functional groups (Scheme 1) ^[13]. These caged luciferins cannot produce bioluminescence until the luciferins (or aminoluciferins) are released or generated via reacting with specific target molecules and subsequently oxidized by luciferase ^[14]. However,it should be emphasized that such an approach is mainly based on the specific reaction-based cleavage of the caged group by targets of interest. Unlike enzyme and small active molecules that can utilize their catalytic or reactive activity to design a turn-on switch,for those biomolecule targets with no catalytic or reactive activity,such as GPCRs,ion channels,DNAs and RNAs,how to develop a turn-on switch for bioluminescence is still challenging. In the field of FLI,a versatile fluorescence off/on approach,named photoinduced electron transfer (PET),is widely used for the fluorogenic probe design ^[15]. In general,PET refers to an excited state electron transfer process by which excited electron is transferred from the donor to acceptor ^[16]. Recently,many PETbased fluorescent probes were designed and utilized to detect the quantity of biomacromolecules,such as GPCRs,ion channels, DNAs,and RNAs ^[2]. Consequently,such a PET-based approach to developing fluorescent probes may give us valuable insights into new rational design strategy of bioluminescence probes to expand the range of application of BLI.

Scheme 1.

	Download: JPG larger image
Scheme 1.Design idea of caged luciferins.

To develop a novel design strategy for bioluminescence probes, Urano and his coworkers at the University of Tokyo recently made their efforts on integrating PET to the design of functional bioluminescent substrates ^[2]. To the best of our knowledge, this report is unprecedented to use different design principles to design functional bioluminescent probes instead of caged luciferin approach. They supposed that luciferase substrates could be similarly developed by PET principle,since the bioluminophore also needs to undergo singlet-excited states through enzymatic reaction like fluorophore. Therefore,to investigate the availability of employing the excited state of a luminophore as a principle for controlling luminescence,they designed and prepared substrate 1 with an electron-donating aniline moiety and substrate 2 with a less electron-donating anilide moiety from the aminoluciferin (AL). The bioluminescence intensity of substrate 2 was about 70-fold higher than that of substrate 1 (Scheme 2),which supports the idea that luciferase-dependent bioluminescence can be modulated by bioluminescent enzyme-induced electron transfer (BioLeT).

Scheme 2.

	Download: JPG larger image
Scheme 2.The design strategy of functional bioluminescence probes based on bioluminescent enzyme-induced electron transfer (BioLeT) and luminescence comparison of substrates 1 and 2 upon reaction with luciferase.

Subsequently,to validate this proof-of-concept,they designed a series of AL derivatives containing benzene moieties with various HOMO energy levels. As the HOMO energy level of the benzene moiety increasing the bioluminescence was quenched,which strongly prove the feasibility of BioLeT as a new design strategy (Fig. 1). Noteworthy,once the HOMO energy level of the benzene moiety was more than -5 eV,the bioluminescence intensity of AL derivatives became very weak. Therefore,the HOMO energy level of the benzene moieties would be an important criterion for whether there is BioLeT in AL derivatives or not. In light of the correlation between luminescence intensity and HOMO energy level,bioluminescent probes with the benzene moiety can be conveniently designed in which HOMO energy level would make a difference upon reaction with biomolecules. However,it should be mentioned that the bioluminescence intensity of these probes depended on the consumption speed of substrate and the quantum yield from the singlet excited state. Compared to the caged bioluminescent probes,the bulkiness and lipophilic groups are available in BioLeT probes,which may be an important factor for the reaction rate of substrate consumption.

Fig. 1

	Download: JPG larger image
Fig. 1Relationship of HOMO energy level of the benzene moiety and luminescence intensity of AL derivatives used for verifying the occurrence of BioLeT.

To further demonstrate BioLeT as a brand new principle for developing new bioluminescence probes,such a group designed and synthesized diaminophenylpropyl-AL (DAL,a novel NO bioluminescent probe). Upon reaction on NO under air,the diaminophenyl moiety with -4.68 eV becomes the benzotriazole moiety with -6.22 eV. As expected,the bioluminescence intensity of DAL presented 41-fold higher than that of DAL-T. These results strongly support the idea that the difference of bioluminescence intensity of DAL and DAL-T should be regulated by BioLeT. They also examined the practicability of DAL as an NO probe in vitro,in cellulo,as well as in transgenic rats ubiquitously expressing firefly luciferase (luc-Tg rat). As a result,there is the positive correlation relationship between the bioluminescence intensity of DAL and concentration of NOC7 (an NO donor) solution. Importantly,the detection limit of DAL is 1.51 mmol/L,which is very suitable for the trace detection of NO. Furthermore,to demonstrate the availability of DAL in cellulo,they incubated HEK293 cells stably expressing luciferase with different concentrations of NOC7 and DAL. Luminescence signal showed a positive linear relationship with the concentration of NOC7 in cellulo. In addition,when injected i.p. DAL (1 mmol) and a freshly prepared NOC7 (20 mmol) solution in PBS,DAL could detect NO released from an NO donor in the peritoneal cavity of luc-Tg rat. These results directly demonstrate that DAL is applicable for BLI in living systems. Therefore,the BioLeT-based strategy can be successfully feasible to design and develop a new class of bioluminescence probe for NO rather than the caged-luciferin approach.

The current work of the Urano group has made a substantial breakthrough in rational design strategies of bioluminescence probes,BioLeT. The combination or binding of probes with targets may lower the HOMO energy level of the quencher or increase the distance between the bioluminophore and the quencher,so as to result in the BioLeT process ‘‘on/off’’. By using this approach,a novel bioluminogenic probe for NO was well developed,which confirmed the availability of BioLeT. It is to be expected that this state-of-the-art BioLeT approach will enable us to design and develop new bioluminescence probes for detecting various biomolecules with no catalytic or reactive activity,such as GPCRs, ion channels,DNAs and RNAs,which will significantly broaden the application of BLI in vitro and in vivo.

Financial support from the National Program on Key Basic Research Project (No. 2013CB734000),the Program of New Century Excellent Talents in University (No. NCET-11-0306),the Shandong Natural Science Foundation (No. JQ201019) and the Fundamental Research Funds of Shandong University (No. 2014JC008).