A simple oxazoline as fluorescent sensor for Zn2 + in aqueous media

    A simple oxazoline as fluorescent sensor for Zn 2 + in aqueous media Weilong Che, Tiecheng Yu, Dan Jin, Xinyao Ren, Dongxia Zhu, Zhongmin Su, Martin R. Bryce PII: DOI: Reference:

S1387-7003(16)30088-0 doi: 10.1016/j.inoche.2016.03.025 INOCHE 6275

To appear in:

Inorganic Chemistry Communications

Received date: Revised date: Accepted date:

23 January 2016 29 March 2016 31 March 2016

Please cite this article as: Weilong Che, Tiecheng Yu, Dan Jin, Xinyao Ren, Dongxia Zhu, Zhongmin Su, Martin R. Bryce, A simple oxazoline as fluorescent sensor for Zn2 + in aqueous media, Inorganic Chemistry Communications (2016), doi: 10.1016/j.inoche.2016.03.025

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ACCEPTED MANUSCRIPT A simple oxazoline as fluorescent sensor for Zn2+in aqueous media Weilong Che,a Tiecheng Yu,b Dan Jin,a Xinyao Ren,a Dongxia Zhu,*a Zhongmin Su,*a Martin R. Bryce*c a

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Key Laboratory of Nanobiosensing and Nanobioanalysis at Universities of Jilin Province, Department of Chemistry, Northeast Normal University, 5268 Renmin Street, Changchun, Jilin Province 130024, P.R. China E-mail:[email protected]; [email protected] b Department of Orthopedics, First Norman Bethune Hospital of Jilin University, Jiefang Road 635, Changchun 130020 P.R.

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China c Department of Chemistry, Durham University, Durham, DH1 3LE, UKE-mail: [email protected]

Keywords：

ABSTRACT

2-(2’-Hydroxyphenyl)-2-oxazoline is shown to be a highly-selective Zn2+

Zinc

sensor with very simple molecular structure. It demonstrates an excellent

Oxazoline

fluorescence “turn-on” response to Zn2+ in aqueous medium even in the

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presence of other competing anions and detection capability for studying

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Fluorescence

the distribution of Zn2+in living human HeLa cells as a proof- of - concept.

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Fluorescent chemosensors continue to attract great

available Zn2+ sensors do not effectively distinguish between Zn2+ and Cd2+. However, the detection of zinc

the simplicity and high sensitivity of fluorescence

has always been problematic due to its inherent d10

signalling.[1] Sensors targeting heavy and transition

shell

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attention for in vitro and in vivo applications because of

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metal (HTM) ions are very important because of the

and

the

characteristics. 2+

[22]

lack

of

spectroscopic

There is an urgent need for improved

current widespread use of these metal ions and their

Zn

subsequent

and improved sensitivity, selectivity and reliability.

pollution

which

environmental and health problems.

triggers

[2]

serious

Zinc, the second

chemosensors with simple molecular structures

Fig. 1 (a) Molecular structure of Hoz. (b) X-ray crystal

most abundant transition metal ion in the human body

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after iron, plays a vital role in many intracellular processes, including gene transcription, regulation of metalloenzymes, neural signal transmission and apoptosis.[3-7] In this regard, more effective detection methods, using selective and versatile sensors are needed for ever increasing applications in biological and environmental science. The design of fluorescent chemosensors for the selective detection of Zn2+ or 2+

Zn /Cd

2+

in an aqueous solution has been considered

as a key target.[8-16] Moreover, a few of the fluorescent methods have been applicable in vivo due to their ultraviolet band excitation wavelength and relative low membrane

permeability.[17-21]Fluorescent

chemosensors also suffer from interference of some HTM ions such as Fe2+, Co2+, Ni2+, Cu2+, Hg2+, and especially Cd2+. Since Cd2+ is in the same group of the periodic table and has similar properties to Zn2+, some

structure of Zn2(oz)4. Hydrogen atoms are omitted for clarity.

We now report a simple fluorescent Zn2+ sensor using the ligand 2-(2’-hydroxyphenyl)-2-oxazoline (Hoz, Fig.1a) that is known as a unit for the construction polymers.

[23,24]

of

blue

luminescent

coordination

Hoz has a simpler conjugated system

compared with other fluorescent chemsensors for Zn2+.[25] In this molecule, the hydroxyphenyl and oxazole groups provide two binding sites (an oxygen

ACCEPTED MANUSCRIPT and nitrogen atom, respectively, which act as good 2+ [26]

donors toward Zn .

Hoz liands.

Based on this consideration,

we have studied Hoz for highly selective and sensitive detection of Zn2+ in aqueous solution and in vivo

The

spectroscopic

characteristics

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experiments using Hela Cells. Zn2+

and

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response of Hoz in physiological conditions were investigated. The titration studies were carried out in HEPES

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CH3CN/aqueous

(2-[4-(2-hydroxyethyl)-1-piperazinyl] ethane sulfonic acid) buffer (1 mM, pH = 7.3; 1:4, v/v). The electronic absorption spectrum of Hoz (10μM) exhibits two sharp = 2.72×103 M–1cm–1) (Fig. 2a). Upon the gradual addition of increasing amounts of aqueous Zn2+

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solutions (0−200 μM) a new absorption peak at 330

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bands at 245 nm(ε = 5.61×103 M–1cm–1) and 304 nm(ε

nm(ε = 2.83×103 M–1cm–1) appeared with the concomitant decrease of the peak at 304 nm(ε = 2.12×103 M–1cm–1). The absorption peak at 330 nm

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shows a significant increment in the absorption

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intensity. It can probably be attributed to the intra-ligand charge-transfer as shown in the theoretical calculation results below. [27]

Fig.2 (a) UV–vis spectra of Hoz (10 μM) upon incremental

446 nm in CH3CN/aqueous HEPES buffer at pH = 7.3

addition of Zn2+ in CH3CN/aqueous HEPES buffer (1 mM, pH

(quantum yield: 0.24) shown in Fig. 3a. Upon the

7.3; 1:4, v/v). (b) Fluorescence emission spectra of Hoz (10

gradual addition of increasing amounts of aqueous

μM) upon addition of Zn2+ in a CH3CN/aqueous HEPES

Zn2+ solutions (0−200 μM) to the solution of Hoz, the

buffer (1 mM, pH 7.3; 1:4, v/v). The Zn2+ concentrations are 0,

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Free Hoz displays a moderately strong emission at

Zn2+ ions caused a blue shift in the emission band to

10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180 and 200 μM,

430 nm and dramatically enhanced the fluorescence

from bottom to top (Ex = 320 nm; slit ex = 10 nm, em = 5 nm).

intensity by about 4.1-fold (Fig. 3a) and the quantum

Insert: The titrtion curve of Hoz reacted with Zn 2+.Each

yield increased up to 0.58. It is shown that the

spectrum was recorded at 430nm.

coordination of Hoz to Zn

2+

is completed within

seconds. The fluorescence response of Hoz to Zn2+ in aqueous solution is visible even with naked eyes.

fluorescence titration experiments, which indicates that the binding stoichiometry between Hoz and Zn

2+

is 1: 2 (Fig. S6, ESI†).The molecular structure of was

also

confirmed

by

X-ray

crystallographic analysis of single crystals, which were obtained by ether diffusion into saturated CH2Cl2–CH3OH (3 : 1 v/v) solutions (Fig 1b). The structure revealed a 1: 2 stoichiometry of Zn

2+

Hoz,

1

equiv

of

a

sodium

salt

of

ethylenediaminetetraacetic acid (EDTA) solution was

The Hill coefficient n was found to be 1:1.8 from

Zn2(oz)4

To investigate the reversible sensing process of

and

added to the solution of Hoz, which was preincubated with 100μM of aZn2+ solution. The initial emission intensity of Hoz was recovered immediately from fluorescent Zn2(oz)4complexes after the addition of an EDTA solution. The high reversibility of Hoz toward Zn2+ complexation and the potential in real-time monitoring is shown in Fig. S5. The detection limit of Hoz as a fluorescent sensor for the analysis of Zn2+ was determined from a plot of normalized fluorescence

ACCEPTED MANUSCRIPT Fig. 3 (a)Fluorescence emission spectra of Hoz (10 μM) in

metal ions (Fig. S7, ESI†) and it was found that Hoz

the presence of Zn2+ and Cd2+ ions in a CH3CN/aqueous

has a detection limit of 0.31μM (45 ppb) for Zn2+. This

HEPES buffer (1 mM, pH 7.3; 1:4, v/v). Insert: Visual

detection limitis comparable to other recently reported

change in the fluorescence of Hoz in presence of Zn2+ and

Zn2+ chemosensors[28] and sufficient to sense Zn2+ ions

Cd2+ ions. (b) Normalised fluorescence emission spectra of

in practical applications.[29]

Hoz in the presence of Zn2+ and Cd2+ ions.

observed upon addition of Zn2+ and Cd2+ to the solution of Hoz. It is notable that Zn2+ caused a significant blue shift and narrowing of the emission profile of Hoz to 430 nm (quantum yield increased to 0.58), while Cd2+ had very little effect on the emission of Hoz (quantum yield 0.20), as shown in Fig. 3. This between Zn2+ and Cd2+ in aqueous solution.

presence of various metal ions. (b) Normalized fluorescence responses of Hoz (10 μM) to cations in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4, v/v). The black bars represent the emission intensities of Hoz in the presence of cations of interest (100 μM). The red bars represent the change in the emission that occurs upon the subsequent addition of Zn2+ to the Hoz-metal cation solutions.

We studied the preferential selectivity of Hoz as a

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difference in response allows Hoz to easily distinguish

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However, selective and fluorescent enhancement was

Fig.4(a) Visual change in the fluorescence of Hoz in the

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significant response in organic fluorescent sensors.

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It is well known that cadmium ions often exhibit a

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intensity as a function of the concentration of the added

fluorescence chemosensor for the detection of Zn2+ in the presence of various competing metal ions: Ag+, Hg2+, Mg2+, Ca2+, Co2+, Pb2+, Ni2+, Cu2+, Cd2+, Cr3+,

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Fe2+ and Fe3+. For these experiments, when 100 μM of Zn2+ was added into the solution of Hoz in the presence of 100 μM of the other metal ions, the emission spectra displayed a similar profile at near 430 nm as with Zn2+ ions only. The emission of Zn2(oz)4 is essentially unperturbed in the presence of these metal ions (Fig. 4a), which indicates that Hoz has the strong affinity and selectivity for Zn2+. It is notable that the addition of Zn2+ to these solutions induced an immediate enhanced fluorescence profile as shown in Fig. 4b except for Cu2+ and Fe3+; this phenomenon is often encountered in many fluorescent sensors.[30] The selective fluorescence enhancement by Zn2+ can be explained as follows: the binding of Hoz to the zinc ion to form [Zn2(oz)4] presumably increases the molecular rigidity and reduces the non-radiative decay pathways for the excited states. These results clearly demonstrate that the Hoz sensor shows a very high selective binding affinity Zn2+ ion even in the presence of other metal ions.To investigate the thermal stabilities of Hoz, thermogravimetric analyses (TGA) was performed in the temperature range of 20–800 ℃ under a flow of nitrogen (Fig. S8, ESI†). Hoz started to decompose at128 ℃, indicating a well stability of Hoz at room temperature.

ACCEPTED MANUSCRIPT fluorescence images of the HeLa cells cultured in the presence of ZnCl2 (5 μM) in DMEM buffer at 37oC

for 15

min; (e) bright-field and (f) fluorescence images of HeLa cells cultured in the presence of Hoz (10 µM) in DMEM

for an additional 15 min.

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buffer at 37 oC

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buffer at 37 oC for 30 min and ZnCl2 (5 µM) in DMEM

The possibility of using Hoz in fluorescence imaging for Zn2+ was studied in living cells using

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Fig. 5 The contours of molecular orbitals of the Zn2(oz)4

scanning confocal microscopy (Fig.6). Human HeLa

complex involved in dominant transitions.

cells were incubated with 10 μM Hoz and 5 μM of Zn2+ ion in Dulbecco's Modified Eagle Medium (DMEM) at

employed the time dependent-density functional

37 °C for 30 min. The results of the bright-field

theory (TD-DFT) with B3LYP exchange-correlation

measurements (Fig. 6a, c and e) suggest that the cells

functional to simulate the absorption spectrum of Zn2(oz)4 complex based on the crystal structure (Fig.

are viable throughout the imaging experiments upon treatment with both Hoz and Zn2+. No intracellular fluorescence can be seen in Fig. 6b, d. However, a

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1b) and optimized ground state geometry. The solvent

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To investigate the fluorescence mechanisms, we

dramatically enhanced intracellular fluorescence is

polarized continuum model (PCM) to model a valid

observed in Fig. 6f after the addition of Zn2+ (5 μM) to

approximation of chemical environment. Besides, the

the cells stained with Hoz, which were incubated for

convergence calculations to 10-6 on the energy and 10-4

another 0.5 h. The marked increase in intracellular

on the wave function were adopted during the

fluorescence suggests that Hoz is membrane permeable

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effect in CH3CN was taken into account using the

and could respond to the presence of Zn2+ in living cells.

the Gaussian 09 program package.The calculated

Thus, Hoz is identified as a potential probe for studying

dominant frontier molecular orbitals (MOs) of the

the distribution and physiological activity of Zn2+ in

Zn2(oz)4 complex are depicted in Fig. 5. As shown in

living

Fig. S9 and Table S1, the calculated lower-energy

proof-of-concept results and further work is needed to

absorption bands are at 325 nm and 308 nm (the

assess if the short wavelength emission may be harmful

experimental values are 330 nm and 302 nm), and

to living cells.

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calculation and all the calculations were performed in

cells.

These

are

promising

initial

contribute to the transitions of HOMO-1 → LUMO+3 and HOMO-2 → LUMO (oscillator strength f = 0.1544 and 0.2545). It is reasonable to assign both of them to intra-ligand charge transfer (ILCT). The higher

energy

band

calculated

at

241

nm

(experimental value at 241 nm) originates from the HOMO−8 → LUMO and HOMO−5 → LUMO transitions (f = 0.3645), which are also intra-ligand charge transfer processes. In this sensor, coordination of zinc ions enhances the fluorescence intensity of Hoz via the chelation-enhanced fluorescence (CHEF) effect, which suppresses the photoinduced electron transfer ]

(PET) quenching process.[31

In conclusion, based on an easily prepared small organic molecule, Hoz, a simple, highly selective and highly sensitive chemosensor for the detection of zinc ions in an aqueous solution and in vivo experiments using Hela cells has been developed. Electronic absorption and fluorescence titration studies of Hoz with different metal ions in a CH3CN/aqueous HEPES buffer (1 mM, pH = 7.3; 1:4, v/v) show a highly selective

binding

cells cultured in the presence of Hoz (10 μM) in DMEM buffer at 37

o

C for 30 min; (c) bright-field and (d)

towards

Zn

ions.

Quantification of the fluorescence titration analysis shows that Hoz can detect the presence of Zn2+ even at a very low concentration of 10 ppb. This simple fluorescence

Fig. 6 (a) Bright-field and (b) fluorescence images of HeLa

affinity

sensor

is,

therefore,

relevant

to

ACCEPTED MANUSCRIPT

reported in this article.The work in China was funded by

NSFC(No.

No.21303012,

51203017, 81172183,

No.51473028

31470932),

the

and key

scientific and technological project of Jilin province (20150204011GX). We would like to thank the support from Jilin Provincial Department of Education. EPSRC funded the work in Durham.

Supplementary data

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Schematic molecular structures, experimental

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W.C. and T.Y. contributed equally to the work

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Acknowledgements

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environmental and biological sciences.

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details and additional NMR spectroscopic data are presented.

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Fig. 1 (a) Molecular structure of Hoz. (b) X-ray crystal structure of Zn2(oz)4. Hydrogen atoms are omitted for clarity.

Fig.2 (a) UV–vis spectra of Hoz (10 μM) upon incremental addition of Zn2+ in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4, v/v). (b) Fluorescence emission spectra of Hoz (10 μM) upon addition of Zn2+ in a CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4, v/v). The Zn2+ concentrations are 0, 10, 20, 30, 40, 50, 60, 80, 100, 120, 140, 160, 180 and 200 μM, from bottom to top (Ex = 320 nm; slit ex = 10 nm, em = 5 nm). Insert: Fluorescence intensity as a function of [Zn2+]/[Hoz].

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Fig. 3 (a)Fluorescence emission spectra of Hoz (10 μM) in the presence of Zn2+ and Cd2+ ions in a CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4, v/v). Insert: Visual change in the fluorescence of Hoz in presence of Zn2+ and Cd2+ ions. (b) Normalised

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fluorescence emission spectra of Hoz in the presence of Zn2+ and Cd2+ ions.

ACCEPTED MANUSCRIPT Fig.4(a) Visual change in the fluorescence of Hoz in the presence of various metal ions. (b) Normalized fluorescence responses of Hoz (10 μM) to cations in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4, v/v). The black bars represent the emission intensities of Hoz in the presence of cations of interest (100 μM). The red bars represent the change in the emission that occurs upon

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the subsequent addition of Zn2+ to the Hoz-metal cation solutions.

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Fig. 5 The contours of molecular orbitals of the Zn2(oz)4 complex involved in dominant transitions.

Fig. 6 (a) Bright-field and (b) fluorescence images of HeLa cells cultured in the presence of Hoz (10 μM) in DMEM buffer at 37

buffer at 37oC

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C for 30 min; (c) bright-field and (d) fluorescence images of the HeLa cells cultured in the presence of ZnCl2 (5 μM) in DMEM

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for 15 min; (e) bright-field and (f) fluorescence images of HeLa cells cultured in the presence of Hoz (10 µM) in

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DMEM buffer at 37 oCfor 30 min and ZnCl2 (5 µM) in DMEM buffer at 37 oCfor an additional 15 min.

ACCEPTED MANUSCRIPT 2-(2’-Hydroxyphenyl)-2-oxazoline is shown to be a highly-selective Zn2+ sensor with very simple molecular

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structure.

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ACCEPTED MANUSCRIPT 2-(2’-Hydroxyphenyl)-2-oxazoline is shown to be a highly-selective Zn2+ sensor with very simple molecular structure. It demonstrates an excellent fluorescence “turn-on” response to Zn2+ in aqueous medium even in the presence of other competing anions and detection capability for studying the distribution of Zn2+ in living human

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HeLa cells as a proof- of - concept.