A synergetic FRET/ICT platform-based fluorescence probe for ratiometric imaging of bisulfite in lipid droplets

Highlights

• A ratiometric fluorescent probe (ZFPy) for rapidly monitoring SO₂ derivatives was developed.

• FRET-based ZFPy displayed larger gap (140 nm) between two emission spectra.

• This is a first synergetic FRET/ICT platform for fluorescence probe.

• ZFPy was applied to image exogenous and endogenous SO₂ derivatives in living Hela, HepG2 and L-O2 cells.

• ZFPy showed lipid droplets-targeted ability (Pearson’s coefficient 0.95).

Abstract

A deep-red emission and lipid droplets-targeted fluorescence probe (named ZFPy) for effective bioimaging of bisulfite was developed from flavone moiety and benzoindole derivative based on intramolecular charge transfer (ICT) and Förster resonance energy transfer (FRET) platform. ZFPy displayed promising fluorescence parameters including bright deep red fluorescence (615 nm), large Stokes shift (205 nm), extended emission window gap (140 nm), high absolute fluorescence quantum yield (4.1%) and stable emission signal output. In addition, ZFPy realized ratiometric fluorescence monitoring for SO₂ derivatives with low detection limit (30 nM), preferable linearity, high sensitivity and selectivity. Interestingly, dual fluorophores (i.e. the donor moiety and 1,1,2,3-tetra-substituent-1H-benzo[e]indol-3-ium iodide moiety) released the same emission band about 475 nm to enhance the emission signal when ZFPy reacted with SO₂ derivatives, to the best of our knowledge, this is the first synergetic FRET/ICT platform for fluorescence probe, which might effectively offer ZFPy a high sensitivity and low detection limit in the detection of SO₂ derivatives. More importantly, ZFPy could image exogenous and endogenous SO₂ derivatives in living HeLa, HepG2 and L-O2 cells with good biocompatibility and photostability. ZFPy also preferred to load on lipid droplets with high Pearson’s coefficient (0.95).

Introduction

Sulfur dioxide (SO₂) derivatives are the most serious pollutant species in environments and organism [1,2]. Abnormal concentration of endogenous SO₂ derivatives in organism could induce some diseases such as cardiovascular diseases, endocrine disorder and even cancer [3,4]. Generally, SO₂ derivatives are endogenously generated during the physiological and pathological metabolism of protein, nucleic acids and lipids [5,6]. For these reasons, it is important to explore reliable technologies for the detection of SO₂ derivative levels in living cells and organisms.

In recent years, fluorescent probes have been applied to evaluate and trace species in living system due to their unique advantages including simplicity, non-invasive visualization, high selectivity and sensitivity [[7], [8], [9]]. A mass of fluorescence probes were reported for the monitoring and imaging of SO₂ derivatives, but most of them depended on the single emission signal output. In comparison with single emission probes, ratiometric probes could realize accurate detection by the ratio of two emission signals, which was effective to relieve the interference from conditions, concentrations and instruments by means of self-calibration [[10], [11], [12]]. To design and develop ratiometric fluorescence probes, intramolecular charge transfer (ICT) [13,14], photo-induced electron transfer (PET) [15,16], excited state intramolecular proton transfer (ESIPT) [17,18] and through bond energy transfer (TBET) [19,20] platforms are usually introduced. Comparing with mentioned above, Förster resonance energy transfer (FRET) platform is one model to develop ratiometric fluorescence probes for the detection and bioimaging of SO₂ derivatives because of high energy transfer efficiency, large Stokes shift and emission window gap [21,22]. FRET-based platform was constructed by a donor, linker and acceptor moiety [23,24]. Such probes could exhibit enhanced emission of the acceptor moiety and quenched emission of the donor moiety when it was excited with excitation wavelength of the donor moiety due to the FRET process from the donor to the acceptor [25,26]. The effective overlap between the emission spectrum of the donor moiety and the absorption spectrum of acceptor moiety in FRET system is essential to increase energy transfer efficiency. Moreover, to ensure detection accuracy, two well-separated emission bands with comparable intensities are crucial. To date, however, the development of practical FRET platform has been retarded by the lack of suitable fluorophore dyad. Therefore, it is a major challenge to tune the spectral overlap and the emission shifts of FRET systems.

Up to now, a number of ratiometric probes for SO₂ derivatives have been reported, but small emission window gap and weak emission signal seriously limits their application [27]. Fluorescence probes with deep-red emission window have received increasing scientific attentions in domains of drug delivery, photodynamic therapy and bioimaging because of their deep tissue penetration, slight auto-fluorescence, less optical damages, low light scattering and absorption [28,29]. In addition, lipid droplets were generated from the aggregation of lipids including cholesterol, triglycerides [30,31]. Lipid droplet was a dynamic subcellular structure for lipid metabolism, while abnormal lipid droplet related to various diseases such as obesity, fatty liver and cardiovascular diseases [32,33]. Unfortunately, few lipid droplet-targeted fluorescence probes have been reported. Therefore, it is very significant to develop a novel ratiometric fluorescence probe for endogenous SO₂ derivatives with high lipid droplet-targeted ability in living cells and organisms.

In this contribution, a flavone derivative was selected as the donor of ZFPy since its good fluorescence, facile preparation and functionalization [34,35]. Besides, flavone derivatives could exhibit yellow or blue fluorescence when they were excited by the light of 350 and 400 nm respectively [36,37]. pH condition also induced clearly changes for the emission band of flavone derivatives since the ESIPT mechanism. More interestingly, the acceptor of ZFPy, 2-(4-(dimethylamino)styryl)-1,1,3-trimethyl-1H-benzo[e]indol-3-ium iodide possessed various advantages, such as good solubility in water, good biocompatibility and high stability [38,39]. Moreover, as the electron-withdrawing moiety in the acceptor, 1,1,2,3-tetramethyl-1H-benzo[e]indol-3-ium (named TBI) could emit intense blue fluorescence [40,41]. So, probe ZFPy with preferable fluorescence factors was constructed based on FRET and ICT platform. The active double bond in the acceptor moiety preferred to react with HSO₃⁻/SO₃²⁻ according to the nucleophilic reaction principle. With the increasing of HSO₃⁻/SO₃²⁻, changes of double emission windows could be recorded, that is the emission signal of ZFPy at 615 nm receded gradually, and the emission signal at 475 nm increased intensively. The ICT process in the acceptor moiety was prohibited and FRET process from the donor to the acceptor turn off. Therefore, the emission of the donor and the emission of TBI moiety increased clearly. Consequently, the extraordinary enhanced signal at 475 nm was derived from the synergetic effect of the same emission band of dual fluorophores (i.e. the donor moiety and TBI moiety), which could effectively offer ZFPy a high sensitivity and low detection limit for the detection of HSO₃⁻/SO₃²⁻.