Article pubs.acs.org/ac
Mitochondria-Targeted Reaction-Based Two-Photon Fluorescent Probe for Imaging of Superoxide Anion in Live Cells and in Vivo Ping Li, Wen Zhang, Kexiang Li, Xiao Liu, Haibin Xiao, Wei Zhang, and Bo Tang* College of Chemistry, Chemical Engineering and Materials Science, Engineering Research Center of Pesticide and Medicine Intermediate Clean Production, Ministry of Education, Key Laboratory of Molecular and Nano Probes, Ministry of Education, Shandong Normal University, Jinan, Shandong 250014, People’s Republic of China S Supporting Information *
ABSTRACT: A newly synthesized reaction-based two-photon (TP) fluorescence imaging probe, 9-butyltriphenylphosphoniumacylamino-2,7-dibenzothiazolineflurene (MF-DBZH), composed of a superoxide anion (O2•−) responsive group and a mitochondria-targeted site, has been shown to have high selectivity toward mitochondrial O2•− fluxes. The fluorescence intensity of MF-DBZH responds proportionally to changes in O2•− concentrations. Moreover, MF-DBZH was proved to be insensitive toward pH changes and has high photostability. Favorable features of this probe also include convenient cell loading, easy staining of both cells and small animals, and excellent biocompibility. Most importantly, MF-DBZH gives reliable TP fluorescent signal to changes of O2•− levels in vivo. background fluorescence due to long-wavelength excitation. In particular, two-photon microscopy (TPM) can offer highresolution, larger depth penetration, as well as less photodamage10,11 in veridical reporting on biologically active molecules. At present, only a few TP fluorescent probes have been achieved to imaging thiols,12 H2O2,13 metal ions,14,15 and so forth; however, TP imaging of O2•− in mitochondria and in vivo has not been reported until now. In this work, we sought to construct a new reaction-based TP fluorescent probe (9-butyltriphenylphosphoniumacylamino-2,7dibenzothiazolineflurene (MF-DBZH)) for imaging O2•− in mitochondria. This probe consists of a fluorene (a potent TP fluorophore), a O2•−-responsive group, and a mitochondrialtargeted site (Scheme 1). Benzothiazoline was utilized as the receptor owing to its good selectivity toward O2•−.16 Triphenylphosphonium salt (TPP+), a well-known potential
M
itochondria, as the evolutionary relics of aerobic bacteria that invaded the proto-eukaryotic cell, play a crucial role in energy metabolism and apoptosis of aerobic organisms. On the basis of plentiful chemical composition, mitochondria are involved in a wide variety of essential physiological processes. Moreover, studies show that mitochondria are the main intracellular source of reactive oxygen species (ROS), an inevitable side product of normal respiration. ROS are responsible for cell signaling and numerous diseases, including cancer, diabetes, and neurodegenerative disorders.1,2 Thus, exploring mitochondrial ROS has received extensive attention, especially with the aid of fluorescent imaging techniques due to their high sensitivity and the satisfying spatial and temporal resolution.3−7 Whether under normal or stress conditions, superoxide anion (O2•−) is first produced within mitochondria, and then rapidly converted to hydrogen peroxide (H2O2). Consequentially, they cause the formations of other ROS,8 which means that O2•− concentrations may reflect the levels of other ROS. Therefore, to develop a novel fluorescent probe for dynamic and specific tracing mitochondrial O2•− has become increasingly significant.5 Furthermore, unraveling the location of O2•− in subcellular compartments can lead to advances in elucidating its chemistry and biology. So far, very few examples of ideal small-molecule fluorescent probes have been reported to distinguish O2•− within mitochondria and in vivo.9 To accurately detect mitochondrial O2•− with appropriate selectivity and sensitivity, background perturbation of abundant biomolecules should be inevitably considered, in view of the complicated biological context in mitochondria. Two-photon (TP) excited fluorescence microscopy associated with the probe label is capable of avoiding © 2013 American Chemical Society
Scheme 1. Reaction between MF-DBZH and O2•−
Received: August 1, 2013 Accepted: September 12, 2013 Published: September 12, 2013 9877
dx.doi.org/10.1021/ac402409m | Anal. Chem. 2013, 85, 9877−9881
Analytical Chemistry
Article
Figure 1. (A) OP fluorescence spectra of MF-DBZH (20 μM) toward various concentrations of O2•− (0, 0.08, 0.4, 0.6, 0.8, 2.0, 3.0, 4.0, 7.0, 8.0, 9.0, 12 μM, from bottom to top). Inset: the linear relationship between fluorescence ratio (F/F0) and O2•− concentrations in the range from 3.0 to 12 μM (probe: 20 μM, Tris−HCl, 10 mM pH 8.0). (B) TP fluorescence spectra for MF-DBZH (20 μM) before (Δ) and after (▲) adding O2•− (12 μM). All of the spectra were acquired in 10 mM Tris−HCl (pH 8.0) at λex = 483 nm (OP) and λex = 800 nm (TP), respectively.
mitochondria-targeted group,2,17 was employed to assist MFDBZH in traversing phospholipid bilayers and accumulating into the mitochondrial matrix driven by the negative membrane potential. We postulated that benzothiazoline dehydrogenation mediated by O2•− resulted in expanding the conjugated system of the sensor molecule. The above reaction process contributes to the TP fluorescence intensity enhancement of MF-DBZH. Herein, we synthesized and characterized the new TP fluorescent probe, and experimental results demonstrated that it not only could effectively monitor mitochondrial O2•− but also successfully image the O2•− fluctuations in mice using intravital TPM.
width of
