Elsevier

Biomaterials

Volume 35, Issue 23, July 2014, Pages 6078-6085
Biomaterials

Live cells imaging using a turn-on FRET-based BODIPY probe for biothiols

https://doi.org/10.1016/j.biomaterials.2014.04.035Get rights and content

Abstract

We designed a red-emitting turn-on FRET-based molecular probe 1 for selective detection of cysteine and homocysteine. Probe 1 shows significant fluorescence enhancement after cleavage of the 2, 4-dinitrobenzensulfonyl (DNBS) unit from the fluorophore upon thiols treatment. The precursor of probe 1, BNM153, is a moderate quantum yield FRET dye which contributes a minimum emission leakage from its donor part. We synthesized this assembly by connecting a low quantum yield (less than 1%) BODIPY donor to a high quantum yield BODIPY acceptor via a 1, 3-triazine bridge system. It is noteworthy that the majority of the non-radiative energy loss of donor (BDN) was converted to the acceptor (BDM)’s fluorescence output with minimum leaks of donor emission. The fluorescence sensing mechanism of probe 1 was illustrated by fluorescence spectroscopy, kinetic measurements, HPLC-MS analysis and DFT calculations. Probe 1 is pH-independent at the physiological pH range. Finally, live cells imaging demonstrated the utility of probe 1 as a biosensor for thiols.

Introduction

Biological thiols such as cysteine (Cys), homo-cysteine (Hcy), and glutathione (GSH) play important roles in a number of biological processes in living organisms [1], [2]. Cys is precursor to the antioxidant GSH, and their abnormal levels are closely related to human ageing and various diseases [3]. While Hcy is a risk factor for Alzheimer's disease and cardiovascular diseases, total plasma Hcy concentrations have been proven to link to birth defects and cognitive impairment at the elderly stage [4]. In view of the importance of thiols, the design of analytical methodologies for the detection of biomolecular thiols in biological and environmental samples has consistently attracted a great deal of attention. Traditional detection methods such as high performance liquid chromatography (HPLC) [5], electrophoresis-based methods [6], as well as electrospray ionization-mass spectrometry [7] have been used to detect thiols. However, these methods are not convenient for use, since they often involve the expensive instrumentation and can be technically demanding in their handling.

On the contrary, fluorescent molecular probes are more attractive, due to their high sensitivity and easy visibility. Also, fluorescent molecular probes offer the possibility of quantitative optical detection of biothiols and broad bioimaging applications, such as detecting the carcinoma region of liver tissue based on the differences of glutathione level [8]. Till now, a number of diverse sensing mechanisms have been employed for thiol detection, including thiol–halogen nucleophilic substitution [9], [10], Michael addition [11], [12], [13], [14], –CHO attached fluorophores [15], [16], [17], [18], and de-protection of 2,4-dinitrobenzenesulfonyl (DNBS)-protected fluorophores [19], [20], [21], [22], [23], [24]. Most of these published probes show good selectivity and sensitivity in detecting certain biothiols by simple spectroscopic techniques, which monitor optical responses caused by different concentrations of biothiols. However, most of them were designed based on a single fluorophore as the fluorescence signalling profile, which may limit their applications. These single fluorophore probes have small Stokes shifts, which can lead to serious self-quenching and fluorescence detection error due to excitation backscattering effects. Some FRET-based probes, constructed with dyes that have high extinction coefficients and high quantum yields as donors, have also been explored for thiols detection. Unfortunately, instances of fluorescence leaks from the donor have been inevitable, as a result of either low energy transfer efficiencies or environmental changes [25], [26].

Hence, newly improved FRET probes with better photophysical properties, which can efficiently eliminate the interference from background fluorescence and scattered light, are worthwhile to design. Several criteria must be satisfied for the FRET probe: 1) absorbance of light at a high extinction coefficient but no fluorescence emission from donor part to ensure that there is no background influence; 2) large pseudo-Stokes shifts and emission shifts to avoid serious self-quenching and fluorescence detection errors caused by excitation backscattering effects [27], [28]; 3) high efficient FRET energy transfer. Here, we designed and synthesized a turn-on FRET-based molecular probe 1 containing a 2,4-dinitrobenzenesulfonyl (DNBS) group on its acceptor, acting as the reaction site for biothiols. In addition, the application of probe 1 for sensing thiols in live cells and its sensing mechanism were also investigated.

Section snippets

Materials

The chemicals and solvents, were purchased from Sigma Aldrich, Acros and Alfa Aesar. All the chemicals were directly used without further purification. Normal phase column chromatography purification was carried out using MERCK silica Gel 60 (Particle size: 230–400 mesh, 0.040–0.063 mm).

Measurements and analysis

HPLC-MS was taken on an Agilent-1200 with a DAD detector and a single quadrupole mass spectrometer (6130 series). The analytical method, unless indicated, is A: H2O (0.1% HCOOH), B: CH3CN (0.1% HCOOH), gradient

Design and synthesis of the probe 1

Here, we designed a reactive FRET probe 1 having long emission wavelength and large pseudo-Stokes shifts characteristics. BDN skeleton was chosen as the donor because of its high extinction coefficient and low quantum efficiency [32]. The red emitting styryl BDM153 was chosen as acceptor part of the FRET pair of probe 1, which is expected to be a good quantum yield BODIPY dye with emission at 580 nm. This elegant choice of donor and acceptor, when successfully incorporated into a triazine-

Conclusions

By introducing the electron-withdrawing group 2,4-dinitrobenzenesulfonyl (DNBS) to the end of the FRET-based BODIPY fluorophore, we constructed a turn-on Cys and Hcy sensor. The unique photophysical properties of precursor of probe 1, such as high ability for light harvesting, no fluorescence leaks from the donor, high quantum yield and large pseudo-Stokes shifts, features attracts us to synthesize probe 1 to be a new candidate for cellular thiols probe. HPLC-MS study and DFT calculations give

Acknowledgements

This study was supported by an intramural funding from A*STAR (Agency for Science, Technology and Research, Singapore) Biomedical Research Council and a Singapore Ministry of Education Academic Research Fund Tier 2 (MOE2010-T2-2-030).

References (38)

  • J. Vacek et al.

    A hydrophilic interaction chromatography coupled to a mass spectrometry for the determination of glutathione in plant somatic embryos

    Analyst

    (2006)
  • Y.Y. Ling et al.

    Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

    Electrophoresis

    (2005)
  • D. Zhai et al.

    A ratiometric fluorescent dye for the detection of glutathione in live cells and liver cancer tissue

    Chem Commun

    (2013)
  • L.Y. Niu et al.

    BODIPY-based ratiometric fluorescent sensor for highly selective detection of glutathione over cysteine and homocysteine

    J Am Chem Soc

    (2012)
  • L.Y. Niu et al.

    A turn-on fluorescent sensor for the discrimination of cystein from homocystein and glutathione

    Chem Commun

    (2013)
  • X.F. Yang et al.

    A seminaphthofluorescein-based fluorescent chemodosimeter for the highly selective detection of cysteine

    Org Biomol Chem

    (2012)
  • X. Zhou et al.

    A sensitive and selective fluorescent probe for cysteine based on a new response-assisted electrostatic attraction strategy: the role of spatial charge configuration

    Chem Eur J

    (2013)
  • M.M. Hu et al.

    Fluorescent chemodosimeter for Cys/Hcy with a large absorption shift and imaging in living cells

    Org Biomol Chem

    (2011)
  • K.S. Lee et al.

    Fluorescence turn-on probe for homocysteine and cysteine in water

    Chem Commun

    (2008)
  • Cited by (90)

    View all citing articles on Scopus
    View full text