fang qian, changli zhang, yumin zhang, weijiang he, xiang gao, ping hu, and zijian guo

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Visible Light Excitable Zn2+ Fluorescent Sensor Derived from an Intramolecular Charge Transfer Fluorophore and Its in Vitro and

in Vivo Application

Fang Qian, Changli Zhang, Yumin Zhang, Weijiang He, Xiang Gao, Ping Hu, and Zijian Guo

J. Am. Chem. Soc., 2009, 131 (4), 1460-1468.

NBD:4-amino-7-nitro-2,1,3-benzoxadiazoleTPEA:N,N,N’-tris(2 -pyridylmethyl)–N’-(2-aminoethyl)- ethane-1,2–diamine

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Zn2+ Plays Vital Roles in Vivo

1. cellular metabolism

2. gene expression

3. Apoptosis

4. neurotransmission

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Schematic Illustration of Putative Zn2+- signaling Pathways

Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 3605–3610.

(1) bind to receptors of ion channels of postsynaptic neurons modulating their a

ctivity.(2) enter postsynaptic neurons via Zn2+- permeable ion channels. (3) be taken back up into the presynaptic neuron and vesicles (4) diffuse away into the extracellular fluid.

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Fluorescence Response of ZnAFs Detecting Extracellularly Released Zn2+ in Hippocampal Slices

J. Am. Chem. Soc. 2005, 127, 10197.

a

ZnAF-2

c

ZnAF-3

d

ZnAF-4

b

ZnAF-2M

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Intact in Vivo Imaging of Ca2+ in Zebrafish Larva

Pfluegers Arch. Eur. J. Physiol. 2003, 446, 766–773.

Maker:Calcium Green-1 AM

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1.They have no UV-induced phototoxicity and autofluorescence

2.They have no sensor-induced interference

What Kinds of Fluorescent Imaging are Essentially Demanded

visible light excited sensor of biocompatibility is appealing

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fluorescence images of fibroblast cells

The Reported Visible Light Excited Zn2+ Fluorescent Sensors

fluorescence images of HeLa cells

(ex)= 400–440 nm (ex)= 356 nm

J. Am. Chem. Soc. 2004, 126, 712 –713

fibroblast cells: 皮膚纖維母細胞 HeLa: 子宮頸癌細胞

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1.visible ICT2.lower interference

R1=NO2, R2=amine(ex)=460~490 nm

SN2

J. Chem. Soc., Perkin Trans. 2 1998, 2165–2173.Biochem. J. 1968, 108, 155–156.

Novel Approach in Developing Visible Light Excitable Zn2+ Fluorescent Sensor

2,1,3-benzoxadiazole (BD)

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Fluorescence Micrographs of Cells Treated with C6-NBD-X (X=PA, PC, PE)

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Chemical Structures of NBD-TPEA, NBD-PMA, and NBD-BPA

ICT absorption and large Stokes shift

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Intramolecular Charge Transfer (ICT)

Donor Acceptorfluorophore

HOMO

LUMO

HOMO

LUMO

h

fluorophore

fluorophore

HOMO HOMO

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Photoinduced Electron Transfer (PET)

Chem. Rev., 2008, 108, 3481-3548.

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Chem. Rev., 2008, 108, 3481-3548.

Photoinduced Charge Transfer (PCT)

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Emission Spectra of NBD-PMA

542 nm(π-π* transition bands)

542 nm→534 nm(PCT effect)

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Emission Spectra of NBD-TPEA

550 nm(ICT transition bands)

542 nm(π-π* transition bands,and blocked PET process)

542 nm→534 nm(PCT effect)

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ICT

π-π*

UV Spectra of NBD-TPEA Obtained during the Titration by Zn(NO3)2

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Emission Spectra of NBD-TPEA Titrated by Zn(NO3)2

Free NBD-TPEA: 550 nmZn2+-NBD-TPEA: 534 nm

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free TPEA

Zn2+:TPEA=0.5:1

Zn2+:TPEA=1:1

1H NMR Spectra of NBD-TPEA obtained during the Titration with Zn2+

☆are for the protons from free sensor and zinc-bound sensor, respectively

19Chem. Commun. 2000, 2395–2396.

Open and filled symbols are for (CF3CO2)2Hgand CF3SO3Ag, respectively. (a) Plots of chemical shifts for protons of fluorophore moiety vs. [Metal ion]/[1]. (□, ■): Ha, (○, ●): Hb

Plots of chemical shifts for protons of the ionophore moiety vs. [Metal ion]/[1].(□, ■) H1, (○, ●) H2, (△, ▲) H3, (◇, ◆) H4, (▽, ▼)H5

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Assignments of 1H NMR Spectra of NBD-TPEA during Zn2+

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Proposed Zn2+ Binding Mode of NBD-TPEA

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Histogram of F/F0 at 544 nm induced by Transition-metal Cations

100 equiv of Na+, Ca2+, Mg2+Zn2+-TPEA Kd=2 nM

Confocal Fluorescence Images of HeLa cells

excited:458 nm

excited:488 nm

NDB-TPEA Zn2+ addition TPEN addition

HeLa: 子宮頸癌細胞 TPEN Kd=9.2×10-14 M

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Confocal fluorescence images of HeLa cells with some makers and NBD-TPEA

mitochondria maker: Red CMXRos

No colocalization

mitochondria : 粒線體

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lysosome maker: (f) LysoTracker Red DND-99 Golgi maker:(k) BODIPY TR ceramide

colocalization

Confocal fluorescence images of HeLa cells with some makers and NBD-TPEA

lysosome: 溶體 Golgi: 高基氏體

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A549: 人類肺腫瘤細胞株

PC12: 上腺髓質嗜鉻細胞瘤 HepG2: 肝癌細胞株

Confocal Fluorescence Images of Some Cells Pre-incubated in ZnSO4

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Confocal Fluorescence Images of PC12 Cells Preincubated with NBD-TPEA

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Something about Zebrafish

1. a common and useful model organism for studies of vertebrate development and gene function.

2. The transparent zebrafish embryo or larva is a widely used model in developmental biology, especially for the study of neurodevelopment.

3. zebrafish from eggs to larvae in under three days.

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Fluorescence Microscopic Images of 4-day-old Zebrafish

Non-stained zebrafish larva

NBD-TPEA-stained zebrafish larva

Zn2+-fed zebrafish larva stained by NBD-TPEA

NBD-TPEA stained zebrafish larva after TPEN

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Fluorescence Microscopic Images of NBD-TPEA-stained Zebrafish Larva

18 h bright-field of (a) 25 h bright-field of (c)

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Fluorescence Microscopic Images of NBD-TPEA-stained Zebrafish Larva

36 h 54 h bright-field of (f)

5-day-old 7-day-old 5-day-old +

TPEN

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Confocal Fluorescence Images of the head (Anterior Lateral-line System) of a 4-day-old Zebrafish Larva

bright-field +

fluorescence

Zoomed bright-field

fluorescence(the left part of

the head)

Anterior Lateral-line: 前側線

fluorescence(the left part of the head)

bright-field +

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Conclusions

1. Novel visible light excited Zn2+ fluorescent sensor, NBD-TPEA, was designed from small ICT fluorophore, ANBD, utilizing its ICT-induced visible ICT absorption and large Stokes shift.

2. Its distinct selective Zn2+-amplified fluorescence and the Zn2+-induced minor emission shift in aqueous medium can be rationalized and attributed to the synergic Zn2+ coordination.

3. In confocal imaging can be used to excite the fluorescence of NBD-TPEA effectively, which facilitates its costaining experiments with other dyes (LysoTracker Red DND-99, BODIPY TR ceramide).

4. Intact in vivo Zn2+ fluorescence and confocal fluorescence imaging of zebrafish larva with NBD-TPEA revealed some interesting phenomena associated with Zn2+ distribution, which appears to have never been observed before.