Author’s Accepted Manuscript A peptide-based fluorescent chemosensor for multianalyte detection Peng Wang, Lixuan Liu, Panpan Zhou, Wenyu Wu, Jiang Wu, Weisheng Liu, Yu Tang www.elsevier.com/locate/bios

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S0956-5663(15)30097-X http://dx.doi.org/10.1016/j.bios.2015.04.094 BIOS7662

To appear in: Biosensors and Bioelectronic Received date: 25 March 2015 Revised date: 28 April 2015 Accepted date: 30 April 2015 Cite this article as: Peng Wang, Lixuan Liu, Panpan Zhou, Wenyu Wu, Jiang Wu, Weisheng Liu and Yu Tang, A peptide-based fluorescent chemosensor for multianalyte detection, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.04.094 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A Peptide-based Fluorescent Chemosensor for Multianalyte Detection

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Peng Wang, Lixuan Liu, Panpan Zhou, Wenyu Wu, Jiang Wu*, Weisheng Liu and Yu Tang**

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Key Laboratory of Nonferrous Metals Chemistry and Resources Utilization of Gansu Province and State Key Laboratory of Applied Organic Chemistry, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou, 730000, P. R. China *

Corresponding Author. E-mail address: [email protected],

**

Corresponding Author. Tel.: 86-931-8912552; fax: 86-931-8912582.E-mail address: [email protected].

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Graphical Abstract

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ABSTRACT

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A novel multifunctional peptide fluorescent chemosensor (DP-3) with a lysine backbone and double sides conjugated with histidine and

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dansyl groups has been designed and synthesized by solid phase synthesis. This chemosensor is a promising analytical tool for detecting

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Zn2+, Cu2+, and S2- based on different mechanisms in 100% aqueous solutions, and intracellular biosensing has been successfully actualized.

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The peptide beacon structure of DP-3 makes it more stable and capable of achieving multianalyte detection, especially for sulfide ions.

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Until now, there have been few examples of using a peptide fluorescent chemosensor to detect anions with a continuous method. As

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designed, DP-3 exhibits excellent cell permeation and low biotoxicity and displays high selectivity and sensitivity, with Zn2+ and Cu2+

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detection limits of 82 nM and 78 nM, respectively. This study raises the new possibility of a highly selective peptide fluorescent

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chemosensor for multifunctional detection, including cation and anions, by different mechanisms in environmental and biological systems.

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We expect that this work will inspire the development of a multifunctional beacon peptide-based fluorescent chemosensor library using

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modifiable lateral and terminal groups for a variety of practical applications in physiological and pathological events.

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Keywords

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Peptide

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Fluorescent chemosensor

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Multianalyte detection

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Biosensing

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Cell imaging

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1. Introduction

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Fluorescent chemosensors are useful tools to detect low concentrations of heavy and transition metal ions (HTM), which are important

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in both environmental and biological systems (Carter et al., 2014; Duke et al., 2010; Guo et al., 2014; Han and Jang, 2014; Iniya et al.,

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2014; Jeyanthi et al., 2013; Ponniah et al., 2014; Sivaraman et al., 2012a; Sivaraman et al., 2014c; Sivaraman et al., 2014d; Yuen et al.,

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2014; Zhang et al., 2011; Zhao et al., 2009). Recent studies have investigated the use of amino acids or short peptides with conjugated

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fluorophores to detect HTM, as these components of metalloproteins can exhibit strong metal-binding affinity, water solubility, and

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biocompatibility (Jang et al., 2012; Lin et al., 2009; Lohani et al., 2012; Ma et al., 2006; Neupane et al., 2013; Neupane et al., 2011; Yang

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et al., 2011). Although various amino acid or peptide based sensors have been developed for metal-ion sensing, multianalyte recognition

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using a single molecule remains a major challenge (Ding et al., 2014; Fu et al., 2014; Goswami et al., 2013; Karakus et al., 2014; Komatsu

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et al., 2005; Lee et al., 2014; Li et al., 2014a; Li et al., 2014b; Maity and Govindaraju, 2012; Rurack et al., 2000; Tayade et al., 2014; Tian

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et al., 2014; Wang et al., 2014a; Zhang et al., 2012). Most multianalyte recognition is focused on sensing cations based on peptide side

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chains or linking fluorophores selectively binding with metal ions. Short peptides that are linear or use a β-turn to build a binding pocket

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for targeting analytes often do not form stable self-assembled “closed” structures (Wu et al., 2012). To conquer this issue, the peptide

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beacon structure DP-3 has been successfully designed to detect multiple analytes, especially sulfides (Scheme 1). Until now, there have

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been few examples of using a peptide chemosensor to detect anions in a continuous method.

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Zn2+, as the second most abundant transition metal ion in the human body, plays a very important role in physiological processes such as

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gene transcription, protein regulation, and signal transmission in neural cells (Komatsu et al., 2007; Li et al., 2014a; Sivaraman et al.,

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2012b; Sivaraman and Chellappa, 2013; Tayade et al., 2014; Vallee and Falchuk, 1993). As the third most abundant transition metal ion in

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the human body, Cu2+ also plays a key role in various biological processes, and its homeostasis is critical for the metabolism and

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development of living organisms (Anand et al., 2014; Lee et al., 2014; Sivaraman et al., 2013; Sivaraman et al., 2014a; Sivaraman et al.,

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2014b; Yin et al., 2014). So, selective detection of Zn2+ and Cu2+ has become attractive due to their fundamental roles in biological and

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environmental processes (Guo et al., 2015; Que et al., 2008; Shen et al., 2014; Wang et al., 2014b). Herein, we present DP-3, which

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contains histidine residue units attached via their C-termini to a central lysine spacer, and each peptide arm is functionalized with a dansyl

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fluorophore moiety at its N-terminus. As the histidine residue is capable of bonding Zn2+ and Cu2+, DP-3 could realize the detection of

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Zn2+ by a turn-on response and Cu2+ by a turn-off response in 100% aqueous solutions. DP-3-Cu, a stable complex, can survive with most

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anions at physiological pH, other than S2-, an important anion in environmental and biological systems (Gao et al., 2013). This

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phenomenon uncovers the important information that signals transduction occurs via the reversible formation and separation of the

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complex DP-3-Cu and CuS (Scheme 1). The dansyl fluorophore has been frequently used in chemosensors for metal ions because it

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includes a sulfonamide group and thus plays a critical role in the recognition of specific metal ions (Li et al., 2009; Ma et al., 2008). In

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addition, the binding of Zn2+ to the His moiety of the DP-3 inhibits photo-induced electron transfer (PET) from the indole moiety to the

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dansyl fluorophore, resulting in a turn-on response, and Cu2+ is a typical ion that leads to decreased fluorescent emissions owing to the

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quenching of the fluorescence by a mechanism intrinsic to the paramagnetic species, resulting in a turn-off response. The results of this

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study indicate that DP-3 exhibits high selectivity to Zn2+ and Cu2+ in environmental and biological systems.

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Scheme 1. A schematic representation of the detection systems of DP-3.

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2. Experimental Section

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2.1. Materials and instruments

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Fmoc–His (Trt)–OH, Fmoc-Lys (Fmoc)-OH, N,N-diisopropylcarbodiimide, 1-hydroxybenzotria-zole, and Rink Amide resin were

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purchased from Top-peptide Co., Ltd. (Shanghai, China). Trifluoroacetic acid (TFA), dansyl chloride, dichloromethane (DCM), ethanol,

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triethylamine, triisopropylsilane (TIS), N,N-dimethylformamide (DMF), piperidine and acetonitrile were purchased from commercial

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suppliers and used without further purification. All of the solvents used were of analytical grade. Stock solutions of the perchlorate or

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nitrate salts of the respective ions (Ca2+, Cd2+, Co2+, Cr3+, Pb2+, Ag+, Mg2+, Cu2+, Mn2+, Ni2+, Zn2+, Na+, Al3+, Fe3+, K+ as perchlorates,

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Hg2+ as a nitrate) were prepared with double-distilled water, which was also used throughout the study.

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Absorption spectra were determined on a Varian UV-Cary100 spectrophotometer. Fluorescence spectra and quantum yields

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measurements were performed on a Hitachi F-4500 spectrofluorometer. The lifetime measurements of the samples were measured using an

12

Edinburgh Instrument FSL920. All measurements were carried out at room temperature. The freeze drying of the sample was performed

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using an FD-1 Ultra-low freeze dryer. All pH measurements were made with a pH-10C digital pH meter. Electrospray ionization MS

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(ESI-MS) spectra were determined on a Bruker Daltonics Esquire 6000 spectrometer. The crude product was purified by HPLC with a

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Vydac C18 column. Cell experiment was operated on Zeiss LSM 710 confocal microscope. All the photographs were taken using a Canon

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digital camera under the illumination of a 365 nm UV lamp.

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2.2. Solid phase synthesis of DP-3

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DP-3 was synthesized using a solid phase synthesis with Fmoc chemistry. Fmoc protected Fmoc-Lys (Fmoc)-OH (0.23624 g, 0.1

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mmol, 4 equiv.) was assembled on Rink Amide resin. After deprotecting the Fmoc group from the resin-bound lysine, Fmoc–His

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(Trt)–OH (0.4958 g, 0.2 mmol, 4 equiv.) was coupled, and dansyl chloride (0.2158 g, 0.2 mmol, 4 equiv.), DMF (6 ml) and triethylamine

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(60 µl) were added. After sequentially washing the resin with DMF (5 ml), DCM (5 ml) and MeOH (5 ml) three times, it was dried using

22

pumps. Cleavage of the peptide from the Rink Amide resin was achieved by treatment with a mixture of 6 ml TFA: TIS: H 2O (95:2.5:2.5,

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v/v/v) at room temperature for 4 h. The crude DP-3 was triturated with diethyl ether chilled at −20◦C and then centrifuged at 5000 rpm for

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5 min at −10◦C. The crude product was purified by HPLC with a Vydac C18 column using a water (0.1% TFA)-acetonitrile (0.1% TFA)

25

gradient to produce overall yield of 76% DP-3. The successful synthesis was confirmed by ESI mass spectrometry, and its homogeneity

26

(>95%) was confirmed by reversed phase analytical HPLC with a C18 column. The DP-3 was characterized by its melting point.

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2.3. General fluorescence measurements

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A stock solution of DP-3 at a concentration of 10−3 M was prepared in distilled water and stored in a cold and dark place. This stock

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solution was used for all fluorescence measurements. All of the detections of the metal ions were from a 100% aqueous phase containing

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10 mM HEPES buffer at pH 7.4. The fluorescence emission spectrum of DP-3 was taken using a 10 mm path length quartz cuvette. The

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emission spectra of the sensor in the presence of various metal ions (Ca2+, Cd2+, Co2+, Cr3+, Pb2+, Ag+, Mg2+, Cu2+, Mn2+, Ni2+, Zn2+, Na+,

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Al3+, Fe3+, and K+ with perchlorate anions and Hg2+ with nitrate anions, prepared in ddH2O) were measured by excitation with 330 nm.

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The slit sizes for excitation and emission were both 5 nm. The concentration of the probe was confirmed by UV absorbance at 330 nm for

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the dansyl chloride group.

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2.4. The binding constants of DP-3-Zn and DP-3-Cu

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The association constants for 2:1 complex were calculated based on the titration curve of the probes with metal ions. Association constants were determined by a nonlinear least squares fitting of the data with the following equation as referenced elsewhere.

y

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x xb  2 2 2  a  b  1  x 

Where x is I-Io/Imax-Io, y is the concentration of metal ions, a is the association constant, and b is the concentration of probe (Neupane et al., 2013).

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1

2.5. The detection limit for Zn2+ and Cu2+

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The detection limit was calculated based on the fluorescence titration. The emission intensity of DP-3 without Zn2+ and Cu2+ was

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measured 10 times, and the standard deviation of the blank measurements was determined. A good linear relationship between the

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fluorescence intensity at 545 nm and the Zn2+ and Cu2+ concentrations could be obtained in the 0-1.25 μM concentration range (R = 0.9986

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and R = 0.9988). The detection limit was then calculated as detection limit = 3σ/k, where σ is the standard deviation of the blank

6

measurements, and k is the slope of the intensity versus sample concentration.

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2.6. Cell culture and cell imaging

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HeLa cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% FBS. All cells were supplemented

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with an antibiotic antimycotic solution (100 units ml penicillin, 0.1 mg ml-1 streptomycin, and 0.25 mg ml-1 amphotericin B) and grown at

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37 ◦C in standard cell culture conditions (5% CO2, 95% humidity). Cell experiment was operated on Zeiss LSM 710 confocal microscope

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with excitation at 405 nm and emission at 545 nm. HeLa cells were attached to the plate 24 h before study.

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2.7. Cytotoxicity assays

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HeLa cells were cultured in culture media (DMEMC, High Glucose) in an atmosphere of 5% CO 2 and 95% air at 37°C. The cells were

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seeded into 96-well plates at a density of 4 × 103 cells per well in culture media, and then 12.5, 25, 50, 100, or 200 μM (final concentration)

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DP-3 was added. The cells were then incubated at 37°C in an atmosphere of 5% CO 2 and 95% air for 24 h. The absorbance of the cells was

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measured by ELISA.

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3. Results and discussion

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3.1. Solid phase synthesis of chemosensor DP-3

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DP-3 was synthesized by a solid phase peptide synthesis with Fmoc chemistry (Scheme S1). Its high purity (>95%) was confirmed by

20

reversed phase HPLC using a C18 column (Fig. S1, Table S1). The successful synthesis of DP-3 was confirmed by ESI mass spectrometry

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(Fig. S2). As DP-3, based on biomolecules, exhibits good solubility in water, the stock solution of DP-3 was prepared in 100%

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double-distilled water (ddH2O), and all fluorescence experiments were carried out in 10 mM HEPES buffer solution at pH 7.4 without any

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co-solvent.

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3.2. Fluorescence response of DP-3 with various metal ions

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As DP-3, based on biomolecules, exhibits good solubility in water, all fluorescence experiments were carried out in 10 mM HEPES

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buffer solution at pH 7.4 without any co-solvent. The fluorescence spectra of DP-3 excited at 330 nm in the presence of 16 metal ions

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(Ca2+, Cd2+, Co2+, Cr3+, Pb2+, Ag+, Mg2+, Cu2+, Mn2+, Ni2+, Zn2+, Na+, Al3+, Fe3+, K+ with perchlorate anions and Hg2+ with nitrate anions,

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prepared in ddH2O) are presented in Fig. 1. Interestingly, DP-3 exhibited exclusive selectivity for Zn2+ and Cu2+ in aqueous solution and no

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fluorescent response to the other metal ions. DP-3 differentiated between these two detected heavy metal ions by response type, exhibiting

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a fluorescence intensity increase triggered by Zn2+ and a decrease in the fluorescence emission intensity with the addition of Cu 2+. The

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addition of Zn2+ resulted in an approximately 5-fold enhancement in signal intensity and a 30 nm blue shift from 545 to 515 nm. Saturation

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of the emission intensity change required approximately 0.5 equiv. of Zn2+. The emission intensity was also observed to decrease

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continuously with the increasing concentration of Cu2+, and the emission peak was completely quenched upon the addition of 0.5 equiv. of

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Cu2+, with a concurrent 30 nm blue shift from 545 to 515 nm. This reduction in the emission intensity may be due to electron transfer (ET)

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from the excited dansyl fluorophore to the coordinated copper ion. DP-3 has a sensitive response to both Zn2+ and Cu2+, which may result

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from the formation of the coordination compounds between DP-3 with Zn2+ and Cu2+. The reasons may be explained by the HSAB

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(Hard-Soft-Acid-Base) theory. The nitrogen of imidozale on the side chain of His belongs to borderline base and Zn 2+ and Cu2+ are

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borderline acids. The binding affinity between DP-3 and Zn2+ or Cu2+ is stronger than the soft acid Cd2+ which has similar properties to

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Zn2+.

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Fig. 1. (a) Fluorescence emission spectra of DP-3 (10 μM) in the presence of various metal ions in 10 mM HEPES buffer solution at pH 7.4. The molar ratio of

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metal/DP-3 is 1:1. Excitation wavelength: 330 nm. (b) The metal ion selectivity of DP-3 at pH 7.4. The molar ratio of metal/DP-3 is 1:1. Excitation wavelength: 330

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nm. (c) The fluorescence emission color changes of DP-3 excited by a UV lamp (365 nm) upon the addition of various metal cations.

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3.3. Fluorescence titration of DP-3 with Zn2+ and Cu2+ and interference test of various metal ions and pH

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The fluorescence responses of DP-3 to Zn2+ and Cu2+ in 10 mM HEPES buffer solution at pH 7.4 are presented (Fig. 2). With an

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increase in the concentration of Zn2+, we observed a nearly 3-fold increase in the emission intensity at the maximum emission and a

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considerable blue shift for the emission maximum from 545 nm to 515 nm. Increasing the concentration of Cu 2+ also resulted in a

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considerable blue shift of the emission maximum from 545 nm to 515 nm but with a decrease in the maximum emission. From the titration

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curve, this value suggests that DP-3 has a sensitive response to both Zn2+ and Cu2+, which may result from the formation of a 2:1 complex

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with Zn2+ and Cu2+.

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Fig. 2. Fluorescence emission spectra of DP-3 (10 μM) following addition of Zn2+ (0–0.75 equiv.) (a) and Cu2+ (0–0.75 equiv.) (b) in 10 mM HEPES buffer solution at pH 7.4. Excitation wavelength: 330 nm.

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To investigate the interference effect of other metal ions on the detection ability of DP-3 for Zn2+, the fluorescence response of DP-3 to

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Zn2+ was measured in the presence of other metal ions except Cu2+ (Fig. 3). Because Zn2+ and Cd2+ have many similar properties, they

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frequently produce similar spectral changes after interacting with chemosensors. Therefore, it would be of great value to develop a

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Zn2+-selective fluorescent chemosensor that is not affected by Cd2+. The Zn2+-dependent fluorescence response of DP-3 was not affected

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by other heavy/transition metal ions, including Cd2+ and Hg2+ (Fig. S3), and the presence of various anions also resulted in no interference

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(Fig. S4). The interference experiments of Zn2+, Cu2+ and S2- were also performed (Fig. S5), and the detection of any one substance is not

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interfered by the existence of other two ions.

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To assess the potential for biological applications of DP-3, we next concentrated on the pH influence on the fluorescence intensity in 10

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mM HEPES buffer solution (Fig. S6). DP-3 exhibited a sensitive response to Zn2+ in the pH range 7–10. At pH lower than 5, the peptide

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chemosensor DP-3 exhibited very weak fluorescence intensity, regardless of the presence or absence of Zn 2+. Previously synthesized

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peptide chemosensor containing dansyl moiety also showed a very weak emission intensity in acidic pH, which was explained by

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protonation of the dimethylamino group (pKa~4) of the dansyl fluorophore. At pH > 6, the intensity difference between DP-3 and

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DP-3–Zn2+ complex increased with increasing pH, however the emission intensity of the peptide chemosensor in the absence of Zn2+ was 5

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not considerably changed. Considering pKa values of His residues, the negative charge of side groups of His residues must increase with

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increasing pH, which might enhance the interactions between the peptide and Zn2+, resulting in the fluorescence increase of the peptide

3

chemosensor in the presence of Zn2+ in this condition. At pH > 10, the intensity of free DP-3 slightly increased with increasing pH. This

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might be due to the deprotonation of the nitrogen atom of the sulfonamide group (pKa~10), which increased the electron density on the

5

naphthyl ring. In addition, the increase of negative charge of the nitrogen atom might promote complexation of DP-3–Zn2+ complex,

6

resulting in the slight increase of intensity at pH > 10 (Neupane et al., 2011). The results of this study confirmed that DP-3 exhibited a

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sensitive response to Zn2+ in the 7–10 pH range, and the fact that the largest DP-3 signal enhancement occurs at neutral pH suggests that

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the sensor is suitable for monitoring Zn2+ contamination in living cells.

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Fig. 3. Fluorescence response of DP-3 (10 μM) in the presence of Zn2+ (1 equiv.) and various additional metal ions (5 equiv.) in 10 mM HEPES buffer solution at pH

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7.4. The black bars represent the addition of an excess of the appropriate metal ion (50 μM) to a 10 μM solution of DP-3. The red bars represent the subsequent

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addition of 10 μM Zn(ClO4)2 to the solution. Excitation wavelength: 330 nm.

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3.4. Binding mode of DP-3 with Zn2+ and Cu2+

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To investigate the binding mode of DP-3 with Zn2+ and Cu2+, the absorbance titrations were performed. As shown in Fig. S7, upon the

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addition of Zn2+ and Cu2+, the absorbance of DP-3 at 330 nm gradually blue-shifted, and simultaneously the absorbance increased, whereas

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the absorbance at 370 nm and 250 nm decreased. The well-defined isosbestic points at 330 nm clearly indicate the formation of only one

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visible active Zn2+ or Cu2+ complex with the ligand DP-3. The binding stoichiometry and binding affinities of DP-3 for Zn2+ and Cu2+ were

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also investigated.

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A Job’s plot analysis was used to determine the binding stoichiometry and indicated that the interaction involves a 2:1 binding of the

20

sensor to Zn2+ and Cu2+ ions in 100% aqueous solution (Fig. S8). Assuming the formation of a 2:1 complex, the association constant of

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DP-3 with Zn2+ was calculated to be 1.20×1010 M-2 (R=0.9963), and the association constant of DP-3 with Cu2+ was calculated to be

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2.13×1011 M-2 (R=0.9950), using a non-linear curve fitting procedure (Fig. S9). The association constant also indicated that DP-3 has

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potent binding affinity to Zn2+ in 100% aqueous solution. The sensitivity of DP-3 for Zn2+ and Cu2+ was calculated on the basis of the

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linear relationships between the maximum emission intensity at 545 nm and the concentrations of Zn2+ and Cu2+. The detection limits for

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Zn2+ (82 nM) and Cu2+ (78 nM) were obtained based on 3σ/k, where σ is the standard deviation of the blank measurements, and k is the

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slope of the plot of the intensity versus the sample concentration (Fig. S10). These results confirmed that DP-3 can be used to detect

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qualitatively low levels of biological or environmental contamination by Zn2+ and Cu2+. The fluorescence quantum yield (ΦF) of DP-3 was

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determined in HEPES buffer solution in both the absence and presence of Zn2+ and Cu2+ with reference to an anthracene standard. A

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quantum yield ΦF =13.61% was observed for DP-3 in the absence of Zn2+ and Cu2+. Following the addition of 0.5 equiv. of Zn2+, the

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quantum yield increased to14.94%, and upon the addition of 0.5 equiv. of Cu2+, the quantum yield decreased to 7.67%. We also perfected

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the fluorescence lifetime experiments (Fig. S11). The lifetime of DP-3 is 3.69 ns, the average lifetime DP-3-Zn is 17.81 ns and the lifetime

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of DP-3-Cu is 3.64 ns. The quantum yields and lifetimes illustrated the mechanism between DP-3 and Zn2+ is chelating fluorescence

33

enhancement fluorescence (CHEF), resulting in a turn-on response. And Cu2+ is a typical ion that leads to decreased fluorescent emissions

34

owing to the quenching of the fluorescence by a mechanism intrinsic to the paramagnetic species, resulting in a turn-off response.

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Furthermore, quantum mechanical calculations were carried out to identify the configurations of the ligand DP-3 and its coordination 6

1

with Zn2+ and Cu2+ ions. Molecular mechanics force field was used for the geometry optimizations, and then single point calculations were

2

conducted using the B3LYP functional (Lee et al., 1988; Becke, 1993). The 6-31G (d, p) basis set was applies for the C, H, O and N atoms,

3

and the LANL2DZ basis set was applied for Zn and Cu atoms. All the calculations were performed using the Gaussian 09 suite of

4

programs (Frisch et al., 2009). The configurations for the complexation of DP-3 with Zn2+ and Cu2+ ions are shown in Fig. S12. It can be

5

seen that the Zn2+ and Cu2+ ions both binds to each DP-3 molecule via two coordination sites (imidazole N) for the 1:2 type complexes I

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and II .

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3.5. Fluorescence response of DP-3-Cu with S2−

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Sulfide is known to react with copper ions to form a very stable CuS species, which has a low-solubility product constant Ksp = 6.3×10−36.

9

It is very exciting and noteworthy that the compound DP-3-Cu could be regenerated by adding S2− to the solution. This regeneration was

10

not observed following the addition of other common anions, including F−, Cl−, Br−, I−, CO32−, PO43−, HPO42−, H2PO4−, P2O74−, NO2−,

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NO3−, ClO3−, ClO4−, SCN−, HSO3−, SO32−, S2O32−, and SO42−. The remarkably selective fluorescent “on” behavior, exclusive to S 2−, was

12

observed after 5 equiv of sulfide ions was added to the DP-3-Cu system, resulting in the regeneration of fluorescence intensity and the

13

maximum emission peak, coupled with a 30 nm red shift of the emission peak from 515 to 545 nm. Thus, these results demonstrate that

14

DP-3-Cu can selectively detect S2−, without interference from the presence of other types of sulfur-containing ions, such as SO32−, SO42−,

15

HSO3−, S2O32−. Further research into the “off−on” property of the chemosensor evaluated the fluorescence enhancement observed

16

following the addition of various amounts of sulfide ions in the presence of 1 equiv of Cu 2+ (Fig. 4). Both the intensity and shape of the

17

emission spectrum of the system closely matched those of compound DP-3, as they both exhibited regeneration of the fluorescence

18

intensity and emission wavelength. This indicated that Cu2+ was released from the DP-3-Cu complex, resulting in the formation of CuS. A

19

considerable blue shift of the emission maximum from 515 nm to 545 nm, with a decrease of the emission maximum, identified the

20

interaction between the sulfide anion and copper (Fig. 4).

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Fig. 4. (a) Fluorescence emission spectra of DP-3-Cu in the presence of various anions in 10 mM HEPES buffer solution at pH 7.4. (b) Fluorescence emission spectra

23

of DP-3 (10 μM) and Cu2+ (10 μM) in 10 mM HEPES buffer solution at pH 7.4 in the presence of different anions (50 μM). The black bars represent the emission

24

intensities of DP-3, and the red bars represent the emission intensities of DP-3 and Cu2+ (10 μM) after the subsequent addition of 50 μM of different anions (1:

25

DP-3-Cu; 2-19: F−, Cl−, Br−, I−, CO32−, PO43−, HPO42−, H2PO4−, P2O74−, NO2−, NO3−, ClO3−, ClO4−, SCN−, HSO3−, SO32−, S2O32− and SO42−; 20: S2−). The molar ratio

26

of anions/DP-3-Cu is 5:1. (c) Fluorescence titration of DP-3 (10 μM) and Cu2+ (10 μM) following the addition of different concentrations of S2− (0−50 μM) in 10 mM

27

HEPES buffer solution at pH 7.4. Excitation wavelength: 330 nm.

28

3.6. Fluorescence imaging in live cells

29

As the chemosensor exhibited a highly sensitive response to Zn2+, Cu2+, and S2- in aqueous solutions at physiological pH, we

30

investigated whether the sensor penetrated live cells and detected intracellular Zn2+, Cu2+, and S2-. Hela cells were incubated with DP-3 (10

31

μM) in DMEM for 30 min at 37°C, following which Zn2+ (10 μM) and Cu2+ (10 μM) were added for incubation for 30 min. Additionally,

32

various concentrations of Na2S (20 μM) were added to DP-3-Cu, and the treated cells were incubated in a culture medium for 30 min at

33

37°C. After being incubated under these conditions, the cells washed three times with PBS, images were taken with a Zeiss LSM 710

34

confocal microscope with excitation at 405 nm and emission at 545 nm. Hela cells incubated with DP-3-Zn initially displayed a strong

35

fluorescence image, but the fluorescence was eliminated in the presence of DP-3-Cu. Fluorescence was subsequently restored after

36

incubation with sulfide, suggesting that DP-3-Cu interacted with sulfide in the cells (Fig. 5). It was found that the fluorescence

37

chemosensor DP-3 has excellent staining capacity and can detect intracellular Zn2+, Cu2+, and S2-. 7

1 2

Fig. 5. Confocal fluorescence images of Hela cells: Bright-field transmission images of Hela cells after incubation with (a) 10 μM DP-3, (d) 10 μM DP-3

3

and 10 μM Zn2+, (g) 10 μM DP-3 and 10 μM Cu2+, (j) 10 μM DP-3-Cu and 20 μM S2− for 30 min at 37°C. Fluorescence transmission images (b, e, h, k) and

4

merged transmission images (c, f, i, l) of Hela cells corresponding to part bright-field transmission images of Hela cells (a, d, g, j), respectively.

5

3.7. MTT assay of DP-3

6

The viability of cells treated with DP-3 over a range of concentrations for 24 h was evaluated using a modified MTT assay (Fig. S13).

7

The results of this analysis demonstrate that DP-3 did not negatively affect the cell viability over the full range of concentrations measured,

8

indicating that DP-3 did not exhibit cytotoxicity and can potentially be used for intracellular detection in biological systems.

9

10

4. Conclusions

11

We present a novel multifunctional peptide fluorescent chemosensor DP-3 as a promising analytical tool for detecting Zn2+, Cu2+, and

12

S2- in 100% aqueous solutions. Compared to other chemosensors (Table S2), the DP-3 showed excellent cell permeation and low

13

biotoxicity, introducing the possibility of highly selective peptide fluorescent chemosensors for a range of multi-detection by different

14

mechanisms in biological systems. We believe this work will inspire the development of a peptide-based multifunctional fluorescent

15

chemosensor library by modifiable lateral and terminal groups for many practical applications in chemical, environmental and biological

16

systems.

17

Acknowledgments

18

This work was financially supported by the National Natural Science Foundation of China (Projects 21471071, 21431002, and

19

21201091), and the Fundamental Research Funds for the Central Universities (lzujbky-2013-59).

20

Appendix A. Supporting information

21

Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2015.xxx

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Highlight

1. A novel peptide fluorescent chemosensor DP-3 has been designed as a promising analytical tool for detecting Zn2+, Cu2+, and S2- in 100% aqueous solution. 2. The chemosensor displays high selectivity and sensitivity with the detection limits of Zn2+ and Cu2+ measured to be 82 nM and 78 nM. 3. The chemosensor showed excellent cell permeation and low biotoxicity to realize the intracellular biosensing. 4. This study introduces the possibility of a highly selective peptide fluorescent chemosensor for a range of multifunctional detection in biological systems. 1 2

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