Biomimetic Sensing System for Tracing Pb2+ Distribution in Living

Jan 21, 2019 - With good cell permeability, GSSH-2TPE can monitor changes in Pb2+ ... nervous system makes children and fetus at a greater risk of lea...
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Biological and Medical Applications of Materials and Interfaces 2+

A biomimetic sensing system for tracing Pb distribution in living cells based on metal-peptide supramolecular assembly Shilang Gui, Yanyan Huang, Yuanyuan Zhu, Yulong Jin, and Rui Zhao ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b19076 • Publication Date (Web): 21 Jan 2019 Downloaded from http://pubs.acs.org on January 22, 2019

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A biomimetic sensing system for tracing Pb2+ distribution in living

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cells based on metal-peptide supramolecular assembly

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Shilang Gui,†,‡ Yanyan Huang,*,†,‡ Yuanyuan Zhu,†,‡ Yulong Jin,†,‡ Rui Zhao†,‡

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† Beijing National Laboratory for Molecular Sciences, CAS Key Laboratory of

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Analytical Chemistry for Living Biosystems, CAS Research/Education Center for

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Excellence in Molecular Sciences, Institute of Chemistry, Chinese Academy of Sciences,

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Beijing, 100190, China

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‡ University of Chinese Academy of Sciences, Beijing, 100049, China

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* Email: [email protected]

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Abstract

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Metal-peptide interactions provide plentiful resource and design principles for developing

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functional biomaterials and smart sensors. Pb2+, as a borderline metal ion, has versatile

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coordination modes. The interference from competing metal ions and endogenous

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chelating species greatly challenges Pb2+ analysis, especially in complicated living

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biosystems. Herein, a biomimetic peptide-based fluorescent sensor GSSH-2TPE was

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developed initiated from the structure of a natural occurring peptide glutathione. Lewis

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acid-base theory was employed to guide the molecular design and tune the affinity and

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selectivity of the targeting performance. The integration of peptide recognition and

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aggregation-induced emission effect provides desirable sensing features, including specific

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turn-on response to Pb2+ over 18 different metal ions, rapid binding and signal output, as

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well as high sensitivity with a detection limit of 1.5 nM. Mechanism investigation

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demonstrated the balance between the chelating groups and the molecular configuration of

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the sensor contributes to the high selectivity towards Pb2+ complexation. The ion-mediated

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supramolecular assembly lights up the bright fluorescence. The ability to imaging Pb2+ in

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living cells was exhibited with minimal interference from endogeneous biothiols, no

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background fluorescence and good biocompatibility. With good cell permeability, GSSH-

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2TPE can monitor changes in Pb2+ levels, biodistribution thus predict possible damage

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pathways. Such metal-peptide interaction-based sensing systems offer tailorable platforms

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for design bioanalytical tools and show great potential for studying the cell biology of metal

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ions in living biosystems.

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Keywords: lead ion, biodistribution, cell imaging, aggregation-induced emission,

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supramolecular assembly

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Introduction

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Lead ion (Pb2+), a highly toxic heavy metal contaminant, poses severe threats to both

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environment and human health worldwide.1,2 With high body absorption efficiency and

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non-degradable nature, lead accumulates and affects almost every organ systems in body,

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including cardiovascular, renal, and reproductive systems.3,4 By mimicking calcium ion,

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Pb2+ can pass the blood brain barrier and exert its most serious damage to central nervous

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system.3,5 The fact that lead interferes with the developing brain and nervous system

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makes children and fetus at a greater risk of lead poisoning.6,7 Although the industrial

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usage of lead declined, the wide applications of lead in agriculture and household

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continuously brings threats of lead exposure.1,8 Given such situation, it is critical to

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develop rapid, selective and sensitive sensors for Pb2+ screening and detection. Monitoring

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Pb2+ in living biosystems would be further beneficial for understanding the molecular

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mechanism of the adverse effects of this ion.

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Tremendous efforts have been devoted to the detection and treatment of Pb2+ in eco-/ bio-

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samples.9-13 During the past decades, fluorescent probes and materials received

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considerable attentions due to their simplicity and sensitivity for analysis.9,14-16 Capable of

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intracellular and in vivo detection, fluorescent sensors show distinct advantage in imaging

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and real-time monitoring various species in biological samples.10,17,18 Particularly, the

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incorporation of metal ion-targeting ligands to signal activatable dyes provides off-on

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and/or ratiometric strategies with high spatial and temporal resolution and in-situ

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quantitation ability.19,20 However, current chemosensors for Pb2+ bioimaging in living

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biosystems with switchable fluorescence are still limited. As the signal switch, Pb2+-

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recognition ligands are central to the selectivity of the sensors, thus the accuracy of the

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detection. Meanwhile, the binding affinity also directly affects the sensitivity of the

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analysis. Therefore, effective design Pb2+-specific fluorescent sensors, especially the

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recognition ligands are of great importance. 3

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Metal-biomolecule interactions, essential for various biological processes, are ubiquitous

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in nature.21-23 These naturally occurring complexes provides plentiful resource and design

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principles for new recognition ligands.24 It has been revealed that coordination bonds are

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frequently adopted by metal ions to bind proteins, peptides, nucleic acids and DNA.25-27

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For Pb2+, different biomolecules, including DNA, DNAzymes and G-quadruplexes, have

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been employed as the targeting moieties to gate the sensing signals.28-32 The biomolecule-

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based sensors show advantage in high biocompatibility, which would be favorable for Pb2+

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detection in biological samples.28 However, the delicate nature and difficulty in preparation

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of macro biomolecules limit their broad applications. Furthermore, Pb2+ has high similarity

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with several divalent ions (such as Ca2+) in structure and chemical property.5,33 As a

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borderline Lewis acid, Pb2+ usually has cross reactions with other metal ions during ligand

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binding.12,34,35 The interference from competing metal ions and endogenous chelating

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species greatly challenge the selectivity and applicability of the sensors for probing Pb2+ in

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complicated living biosystems.

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Peptides with optional building blocks, rich coordination chemistry and high stability are

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attractive candidates for tailoring selective metal-binding ligands.36-39 In organisms,

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peptides act as ion transporters or scavengers via chelating various metal ions.40,41 Their

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high biocompatibility, ease of chemical synthesis and functionalization further bring

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favorable features for probing species in biological systems.36,42,43 Initiating from the

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structure of an endogenous peptide glutathione (-GSH), herein a biomimetic Pb2+

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recognizer was developed. Based on Lewis acid-base theory, the modification in the

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molecular structure of GSH was expected to tune the non-specific and broad-spectrum

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binding to highly specific towards Pb2+. The incorporation of an aggregation-induced

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emission (AIE) fluorophore44-46 serves as the indicator for the recognition event, while the

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targeting peptide plays a role as the signal switch. The Pb2+-sensing performance was

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assayed with ion-induced fluorescence response, molecular assembly behavior, binding

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kinetics and thermodynamics. Mechanism investigation demonstrated the particular 4

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molecular configuration of the sensor during complexation contributes to the high

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selectivity towards Pb2+. The feasibility of this peptide-based sensor for monitoring cellular

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Pb2+ exhibits its great potential for studying the cell biology of this metal ion in living

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biosystems.

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Experimental

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Synthesis of GSH-TPE. The tetraphenylethylene (TPE) conjugated GSH (GSH-TPE) was

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synthesized with standard FMOC solid phase peptide synthesis strategy (Figure 1). Fmoc-

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Gly-Wang resin was used as the starting material. Piperidine (20% in DMF), 4-

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methylmorpholine and HBTU were used as deprotection reagent, activating reagent, and

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coupling

agent,

respectively.

After

peptide

elongation,

2-(4-(1,2,2-

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triphenylvinyl)phenoxy)acetic acid (TPE-COOH) was conjugated to the amino terminals

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of the tripeptides under the same conditions. Finally, GSH-TPE was cleaved from the resins

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with a freshly prepared reagent (94% TFA, 2.5% H2O, 2.5% EDT and 1% TIPS as

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scavengers). The product was purified on a semi-preparative HPLC column (Dikma-C18,

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250 mm×10 mm i.d.) using water with 0.1% TFA as the aqueous phase and CH3CN with

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0.1% TFA as the organic phase.

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Synthesis of GSSH-2TPE. GSSH-2TPE was obtained by oxidation of GSH-TPE with

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iodine. A beaker with magnetic stirrer was charged with GSH-TPE (30.05 mg) and a

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mixture of water-acetonitrile (4 mL, 1:1, v/v). Then, iodine (5.32 mg) was added at room

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temperature. The reaction was monitored by HPLC. With 24-h oxidation, the mixed

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solution was separated on a semi-preparative HPLC column to collect the product. After

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purification, GSSH-2TPE was obtained as white solid.

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Fluorescence measurement. The stock solutions (0.25 mM) of each metal ions (Pb2+, Al3+,

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Cu2+, Ag+, Ni2+, Li+, Mg2+, Co2+, Ca2+, Cr3+, K+, Na+, Fe3+, Ba2+, Cd2+, Zn2+, Mn2+, Hg2+

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Fe2+) were prepared with ultra-pure water, respectively. A stock solution of GSSH-2TPE

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was prepared by dissolving the solid with DMSO to a concentration of 1 mM. For a typical 5

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detection, 2.5 L GSSH-2TPE stock solution was mixed with the stock solution of metal

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ion (10 L), followed by the dilution with appropriate amounts of water and acetonitrile to

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a final volume of 500 L. Without further incubation, the corresponding fluorescence

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spectrum was recorded with the excitation wavelength of 330 nm. The emission was

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collected from 350 to 625 nm.

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Electron microscopic characterization. The Pb2+-induced GSSH-2TPE assembly was

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characterized scanning electron microscopy (SEM). For sample preparation, the mixed

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solution of Pb2+ (5 μM) and GSSH-2TPE (5 μM) was dipped on cover slips. After dried

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under atmosphere, the sample was coated with a thin layer of gold, then subjected for SEM

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observation.

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Fluorescence imaging of Pb2+ in living cells. HeLa cells were seeded into glass bottom

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culture dishes at a density of 1.0105 cells mL-1 and cultured overnight for adhesion. The

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adherent cells were washed with stroke-physiological saline solution three times and

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loaded with Pb2+ (in stroke-physiological saline solution) at 37 oC for 30 min. After

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removal of free Pb2+ by washing the cells with stroke-physiological saline solution, GSSH-

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2TPE (20 M in stroke-physiological saline solution) was added. After incubated for 30

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min. Fluorescence imaging was performed on an Olympus IX83 Fluorescence microscope

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(Tokyo, Japan) with the USH-1030L 100W high pressure mercury burner (Olympus, Japan)

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as the excitation source. The objective used for imaging was a UPLSAPO 100 oil-

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immersion objective NA 1.40 (oil) (Olympus). Excitation filter: BP325-375 (Olympus),

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emission filter: BA420 (Olympus). Image processing and analysis were performed on

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Olympus software (cellsens standard). For comparison, the imaging parameters of the

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microscope were set to be the same for all samples. Pixel intensity was analyzed on Image-

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Pro Plus software.

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Cytotoxicity assay. HeLa cells were seeded in 96-well plates at a density of 7000 cells per

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well. After 24 h incubation, the culture medium was replaced by GSSH-2TPE solutions at

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various concentrations (2, 5, 10 or 20 m), respectively. The cells were further cultured at 6

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37 oC for another 24 h. The solutions were then discarded, and freshly prepared MTT

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solution (100 L, 0.5 mg/mL in culture medium) was added to each well. This was

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followed by incubation at 37 oC for 4 h. After removal of the MTT medium solution,

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DMSO (150 L) was added to each well, and the plate was shaken for 10 min to dissolve

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the formed precipitates. The absorbance values of the wells were read with a microplate

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reader at 490 nm (Molecular Device, California, USA). The cell viability rate (VR) was

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calculated from the equation VR=A/A0100%, in which A is the absorbance of the probe-

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treated cells and A0 is the absorbance from cells without treatment. All data were obtained

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from three separate experiments.

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Results and Discussion

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Design and synthesis.

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Glutathione (-Glu-Cys-Gly, GSH), an important tripeptide, is well-known for its affinity

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to various metal ions, including Hg2+, Cu2+, Pb2+, Ag+, etc.41 The co-existence of thiol and

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carboxylic acid groups, as well as their specific intramolecular distribution account for the

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binding towards metal ions. Aiming at specific detection of Pb2+, peptide design was started

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from the structure of GSH. As a soft Lewis base, thiol group takes the dominant role for

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the recognition of soft Lewis metal ions, such as Hg2+ and Ag+ (Figure 1). Accordingly,

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carboxyl group as a hard Lewis base preferentially binds hard Lewis metal ions, such as

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Al3+. For Pb2+, as a borderline Lewis metal ion, its property is intermediate between soft

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and hard acids. Therefore, it is important to balance the affinity between thiol and carboxyl

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groups for the development of Pb2+-specific ligand. In this effort, oxidation of GSH to

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GSSH was proposed (Figure 1). The conversion of thiol group to disulfide group was

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supposed to regulate the affinity and selectivity of the peptide. The unchanged molecular

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configuration and remaining carboxyl groups were expected to maintain the binding

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towards Pb2+. 7

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Figure 1. Design, synthesis and sensing concept of GSSH-2TPE for Pb2+ detection. Images

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were taken under UV light (360 nm).

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As the signal reporter, an AIE fluorophore tetraphenylethylene (TPE) was introduced for

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the conjugation with GSSH. The ion recognition-induced fluorescence turn-on effect is

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appealing for highly sensitive sensing. Taking advantage of the high efficiency of solid

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phase synthesis approach, the building unit GSH-TPE was facilely prepared. Direct

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oxidation of GSH-TPE gave the final product GSSH-2TPE (Figure 1). The synthesized

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compounds were characterized with HPLC, MS and NMR (Figure S-1~S-3, Supporting

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Information).

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Tuning ion-induced fluorescence behavior.

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To validate the design and working concept, the fluorescence of both GSH-TPE and

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GSSH-2TPE in the presence of soft (Ag+ and Hg2+), hard (Al3+ and Cr3+) and borderline

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(Pb2+ and Cu2+) metal ions were recorded, respectively. With native GSH as the recognition

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ligand, GSH-TPE responded to all three kinds of metal ions (hard, soft and borderline metal

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ions) with blue fluorescence (Figure 1). Slightly stronger emission was detected for Ag+,

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which was almost probably due to the strong coordination of thiol group towards soft Lewis 8

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metal ions. In comparison, GSSH-2TPE showed distinctively different fluorescence

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performance. Pb2+ brought the highest emission from GSSH-2TPE. No fluorescence was

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detected for GSSH-2TPE solutions added with Ag+ or Hg2+. These results demonstrate that

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the transformation of thiol group to disulfide bond successfully tunes down the affinity

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towards soft Lewis metal ions. The balance between the soft and hard bases leads to the

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recognition of Pb2+. It is also notable that the fluorescence from the binding of Pb2+ to

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GSSH-2TPE is the brightest among all the complexes from GSH-TPE and GSSH-2TPE

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(Figure 1).

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Encouraged by the above results, the fluorescence turn-on behavior of GSSH-2TPE was

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further examined with 19 different metal ions (Figure S-4). Pb2+ induced the strongest

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fluorescence from GSSH-2TPE over the other ions, indicating the selectivity of GSSH-

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2TPE towards Pb2+. It is noticed that after the oxidation of thiol group, the coordination

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effect from carboxyl group becomes prominent. Therefore, binding of GSSH-2TPE to hard

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Lewis metal ions needs to be considered. Among these hard metal ions, only Al3+ brought

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weak fluorescence (Figure 1, Figure S-4). For Al3+, it belongs to p-block elements as Pb2+,

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thus has flexible coordination geometry. Together with its hard Lewis acid property, Al3+

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is able to bind carboxyl groups in GSSH-2TPE. However, the significant difference in ionic

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size leads to different binding affinity. With a Shannon radii of 119 pm, Pb2+ fits the cavity

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of GSSH-2TPE created for chelating. The match in geometry reinforces the coordination

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bonding between Pb2+ and GSSH-2TPE and results in strong fluorescence. The small Al3+

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(53 pm) cannot accommodate the coordination cavity formed by GSSH-2TPE. Although

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there are multiple carboxyl groups in GSSH-2TPE, the mismatched size and loose binding

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between GSSH-2TPE and Al3+ result in weak fluorescence. In biosystems, Ca2+ is usually

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substituted by Pb2+, however Ca2+ cannot mimic Pb2+ to coordinate with GSSH-2TPE

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(Figure S-4). These can be ascribed to the restricted coordination number (6–8) of Ca2+

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which cannot be meet by the structure of GSSH-2TPE (four carboxyl groups). Moreover, 9

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alkali and alkaline earth metal ions tends to form hydrated structure with H2O,47 which

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further prevents it from binding with GSSH-2TPE.

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To address possible interference from Al3+, further regulation of the affinity and selectivity

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of GSSH-2TPE was performed. Ammonium fluoride (NH4F) was employed as the

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competing reagent to adjust the interactions (Figure S-5, Supporting Information). With

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optimized concentration (Figure S-5), NH4F effectively inhibits the Al3+-induced signal by

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blocking its binding to GSSH-2TPE with strong hard-acid bonding of F-. No sacrifice in

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the Pb2+-induced fluorescence was observed. Under these conditions, 18 metal ions with

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different valence and properties were mixed with GSSH-2TPE either individually or as a

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mixture. None of these metal ions can turn on the emission of GSSH-2TPE (Figure 2a).

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Even these borderline metal ions (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) also cannot light up the

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fluorescence. This can be ascribed to their small and unfitted sized for ligand complexation

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(Table S-1), which disabled stable binding with GSSH-2TPE. Furthermore, these transition

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metal ions belong to d-block elements. Their inflexible geometry cannot accommodate

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different ligand configuration.48 Pb2+ is the only metal ion that can induce the blue

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fluorescence signal from GSSH-2TPE, thus can be easily discriminated either individually

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or co-existing with various metal ions in complicated samples. By tuning the affinity with

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molecular design and competing reagent, GSSH-2TPE provides high selectivity for Pb2+

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recognition and detection.

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Figure 2. (a) Fluorescence variation of GSSH-2TPE responding to 19 different metal ions,

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respectively. Insert: fluorescence spectra of GSSH-2TPE in the mixtures of metal ions. Ion

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mixture = Ni2+, Hg2+, Li+, Mg2+, Cu2+, Co2+, Ca2+, Cr3+, K+, Na+, Fe3+, Al3+, Ag+, Ba2+, 10

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Cd2+, Zn2+, Mn2+, and Fe2+. [GSSH-2TPE] = 5 M, [metal ion] = 5 M. (b) Fluorescence

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spectra of GSSH-2TPE (5 M) responding to various concentrations of Pb2+. (c) Plot of

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fluorescence intensity of GSSH-2TPE to Pb2+ concentration. Solvent: aqueous solution of

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NH4F (80 M) containing 20% CH3CN.

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Rapid and sensitive fluorescence turn-on detection of Pb2+.

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Binding kinetics and thermodynamics are crucial for the sensing performance of GSSH-

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2TPE. Kinetic study was performed by real-time monitoring the fluorescence from GSSH-

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2TPE upon the addition of Pb2+ (Figure S-6, Supporting Information). Within 1 min, the

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emission reached the maximum. Such fast binding and signal responding are appealing for

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rapid and real-time analysis.

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Fluorescence titration shows the dependence of the emission intensity on Pb2+

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concentration in the range of 0.1 - 5 M (Figure 2b). The signal obtained a plateau with

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1.0 equiv of Pb2+, indicating the saturation of the binding. According to the 3 rule,49,50

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the detection limit was calculated as 1.5 nM (Figure S-7), which is lower than many

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previously reported fluorescence sensors.29,37,51 This sensing performance can well satisfy

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the requirement of EPA drinking water regulation of Pb2+ (48 nM). From the fluorescence

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binding isotherm, the dissociation constant (KD) between GSSH-2TPE and Pb2+ was

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estimated as 2.1  10-6 M (Figure S-8). Such high affinity contributes to the high sensitivity

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of the peptide sensor.

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In previous works, higher sensitivity has also been reported for some Pb2+ sensors in

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solutions by using nanomaterials and/or DNAzyme.52-55 However, these sensors cannot be

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applied for analyzing Pb2+ in living cells. Given the high biocompatibility and tailorable

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features of peptide, peptide-based sensors attract increasing interests. Among these, GSH

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is the most widely used peptide for Pb2+ sensing. However, the selectivity and sensitivity

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are limited due to its general affinity towards various metal ions. Herein, by taking 11

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advantages of peptide recognition and Pb2+-induced emission effect, GSSH-2TPE shows

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high affinity, selectivity and sensitivity for Pb2+ in biosystems.

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Investigation of Pb2+-peptide assembly mechanism.

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For AIE fluorophores, the switch-on of fluorescence is usually accompanied with the

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formation of nanoassemblies. In the effort to reveal the Pb2+-induced fluorescence

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mechanism, GSSH-2TPE solutions with Pb2+ were characterized with dynamic light

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scattering (DLS) analysis. As shown in Figure 3a, the presence of Pb2+ resulted in the

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generation of nanoparticles with an average diameter of 118 nm. Such phenomenon was

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further confirmed by SEM observation. For GSSH-2TPE only, no obvious assembly was

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found (Figure 3b). Nano-sized particles (~100 nm) were detected in the mixed sample of

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GSSH-2TPE and Pb2+. The emissive behavior of these nanoparticles was imaged with

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fluorescence microscopy (Figure 3f). These results manifest that Pb2+ is the inducer of

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GSSH-2TPE assembling. The generated nanoaggregates are the source of the blue

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fluorescence.

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Figure 3. (a) DLS characterization of the assembly behavior of GSSH-2TPE (5 M) in the

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presence of Pb2+ (5 M). (b) SEM images of GSSH-2TPE without and with the presence

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of Pb2+. Scale bar: 100 nm. (c) MS characterization of the coordination complex from

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GSSH-2TPE and Pb2+ (ESI, negative mode). The calculated m/z for [M + Pb - 4H]2− is

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795.82; the calculated m/z for [M-2H]− is 693.22. (d) FT-IR chemical imaging microscopic

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characterization of GSSH-2TPE (5 M) with and without Pb2+ (5 M). (e) Possible process

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for the formation of coordination complex and supramolecular assembling of fluorescent

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nanoaggregates from GSSH-2TPE and Pb2+. (f) Fluorescence microscopic image of the

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nanoparticles assembled from GSSH-2TPE and Pb2+. Scale bar: 5 m.

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To investigate the molecular basis of the ion-induced assembly, the coordination complex

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was characterized with mass spectrometer, Job’s plot assay, competitive binding assay and

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Fourier transform infrared spectroscopy (FT-IR). In MS spectrum, an m/z signal (m/z

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795.92) attributed to the complex of [GSSH-2TPE + Pb2+] was detected (Figure 3c).

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Binding assays by continuous variation of Pb2+ mole fraction gave a peak at a mole fraction

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of 0.5 in the Job’s plot (Figure S-9). Both results exhibit that the binding stoichiometry of

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GSSH-2TPE with Pb2+ is 1:1. The competition assay with GSSH demonstrates that the

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peptide structure plays the central role for Pb2+ recognition (Figure S-10). The TPE parts

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mainly act as the signal generator. From microscopic FT-IR characterization (Figure 3d),

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significant diminish of the carboxylic C=O vibration signal (1726 cm-1) and the shift of

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stretching vibration of COO- (1414 cm-1) towards lower wavenumber (1409 cm-1) suggest

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the involvement of carboxyl group in GSSH-2TPE for Pb2+ coordination. The shift of

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vibration of amide bonds from 1647 cm-1 to 1638 cm-1 can be ascribed to the formation of

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hydrogen bonds during the formation of nanoaggregates. These results provide useful

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insights into the formation of coordination complex and supramolecular assembly.

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The specificity of GSSH-2TPE towards Pb2+ can be explained with the properties of ligand

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and metal ions. With multiple coordination sites, GSSH-2TPE binds Pb2+ with a 13

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stoichiometry of 1:1, thus is a polydentate ligand. For polydentate ligands, the selectivity

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and stability of the chelation is determined by not only Lewis acid-base theory, but also the

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geometry and cavity size of the coordination complex. As a borderline metal ion, Pb2+

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binds both hard and soft donor atoms, such as oxygen and sulfur atoms in GSSH-2TPE. In

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GSSH-2TPE, there are four free carboxylic acid groups available for Pb2+ chelating (Figure

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3e). The high tendency of Pb2+ to form strong bonds with sulfur-containing groups

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reinforces the stability of the coordination complex. Particularly, the large size of Pb2+ (119

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pm) could also contribute to the selective binding, which well fit the cavity created by the

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ligand. Moreover, as a p-block elements, Pb2+ has versatile coordination chemistry and can

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adopt various geometries. The match in chelating bonds, size and configuration leads to

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the high affinity and specificity between GSSH-2TPE and Pb2+.

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For the signal generation, restriction of intramolecular rotation lays the fundamental for the

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light up of AIE fluorophores. The formation of complex between GSSH-2TPE with Pb2+

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brings steric hindrance which impedes the bond rotation in TPE moieties. Furthermore,

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during the supramolecular assembly of nanoaggregates, the 1:1 complex of GSSH-2TPE

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and Pb2+ is employed as the building block (Figure 3e). The intermolecular noncovalent

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forces including hydrogen bonds between amide and carboxyl groups, - stacking

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between the benzene rings in TPE moieties and electrostatic forces drive the self-

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assembling of nanoparticles. The restriction of intramolecular rotation of TPE structure

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gives rise to the bright blue emission (Figure 3f).

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Imaging detection of Pb2+ in living cells.

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To apply GSSH-2TPE for tracking Pb2+ in living cells, possible interference from

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endogenous biothiol species such as GSH, cysteine, homocysteine needs to be excluded.

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No obvious inhibition to the Pb2+-induced fluorescence signal from GSSH-2TPE was

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detected, when using these biothiol species and their mixtures as competitors (Figure S-

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11). The presence of Ca2+ and high concentration Mg2+ in cells was also considered. No 14

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interference from Mg2+ and Ca2+ was detected even when Mg2+ concentration was as high

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as 1 mM (Figure S-12). These results provides the basis for applying GSSH-2TPE for

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sensing Pb2+ in cells.

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The feasibility of GSSH-2TPE for cell imaging was probed by treating HeLa (cervical

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cancer cell line) cells with and without Pb2+. In the absence of Pb2+, no fluorescence emitted

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from GSSH-2TPE-incubated HeLa cells (Figure 4a). No background and non-specific

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fluorescence were detected, which is favorable to acquire high signal-to-noise ratio and

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high sensitivity. This also further excludes the interference from endogenous Ca2+ and

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Mg2+. In contrast, distinct fluorescence was observed for Pb2+-preloaded cells after the

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incubation with GSSH-2TPE (Figure 4a). These results suggest the capability and

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specificity of GSSH-2TPE for Pb2+ sensing even in complicated biosystems.

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Figure 4. (a) Fluorescence microscopic images of living HeLa cells with GSSH-2TPE (20

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M) with and without the addition of Pb2+ (20 M). (b) Monitoring binding kinetics of

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GSSH-2TPE to Pb2+ in cells. Cells were preloaded with Pb2+ (20 M), followed by the

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addition of GSSH-2TPE (20 M). Images were obtained with different incubation time

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with GSSH-2TPE. Scale bar: 10 m. (c) Pixel intensity analysis of the cell images with

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different GSSH-2TPE incubation time.

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When exists individually, Al3+ induces weak fluorescence of GSSH-2TPE, which may

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bring selectivity problem during biological application. In competitive binding assays,

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Pb2+-responding fluorescence was not affected by the co-existence of Al3+ even at high

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Al3+ concentration (Figure S-13, Supporting Information). Moreover, Al3+ cannot induce

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any fluorescence in cells due to its weak affinity with GSSH-2TPE (Figure S-13d). These

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results ensure the high specificity of GSSH-2TPE for Pb2+ analysis in complicated

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biosystems. To assess the sensitivity of GSSH-2TPE for Pb2+ in cells, lower Pb2+

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concentration were used for the treatment of cells (Figure S-14). At a concentration as low

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as 100 nM, obvious fluorescence still can be clearly observed in cells, while cells without

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Pb2+ treatment remained dark under the same conditions

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Cellular binding kinetics of GSSH-2TPE towards Pb2+ was investigated by monitoring the

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fluorescence of cell images obtained at different incubation times (Figure 4b, c and Figure

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S-15). With 5-min incubation, cell membrane became fluorescent, indicating the binding

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of GSSH-2TPE to membrane-anchored Pb2+. As the incubation time prolonged to 10 min,

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blue fluorescence inside cells was detected, suggesting the internalization of GSSH-2TPE

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and its binding to intracellular Pb2+. With 15-min incubation, the fluorescence of the cells

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further intensified. The signal intensity almost kept the same after treated for 20 min,

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demonstrating the saturation of the binding between GSSH-2TPE and Pb2+. Based on these

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results, 20 min was chosen as the optimal incubation time to reach the binding equilibrium.

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Encouraged by the high sensitivity, selectivity and fast response, GSSH-2TPE was applied

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to trace the dependence of Pb2+ biodistribution on exposure dosage. At a Pb2+ concentration

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of 5 M, blue fluorescence was observed both inside and surrounding the cells (Figure 5b).

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Noticeably, much stronger emission was from cell membrane, denoting that at lower

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concentration, Pb2+ ions tend to be retained by the lipid structure. This is in accordance

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with the membrane association property of Pb2+.56 For HeLa cells loaded with 10 M Pb2+,

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the overall fluorescence signal greatly enhanced. Besides, the fluorescence distribution 16

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pattern also changed. Cytoplasm became strongly emissive with unevenly distributed

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fluorescence. The signal intensity of cytoplasm became similar with that from cell

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membrane. (Figure 5c, e). Co-staining assays with commercial membrane tracker was

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performed to verify and differentiate the signal from cell membrane (Figure S-16).

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Compared with cytosol, cell nucleus displayed weaker fluorescence, indicating lower

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tendency of Pb2+ to enter nucleus. Further increase of Pb2+ loading to 20 M did not change

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the fluorescence intensity and distribution (Figure 5d, e). By locating to a variety of

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subcellular units, Pb2+ interferes with enzyme functions, poisons cellular activity and

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destroy biological systems.

379 380

Figure 5. (a-d) Fluorescence microscopic images of living cells with GSSH-2TPE (20 M).

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(a) HeLa cells only treated with GSSH-2TPE. (b-d) Pb2+-preloaded cells treated with

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GSSH-2TPE. [Pb2+] = 5 M (b), 10 M (c), and 20 M (d). Scale bar: 10 m. (e)

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Subcellular distribution of Pb2+ responding to exposure dosage based on pixel intensity

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analysis.

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Cellular distribution pattern of Pb2+ in different cell lines were examined, including MCF-7

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(human breast cancer cell line), HepG2 (hepatoblastoma cell line) and HEK293 (from

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normal human kidney). Although with different origins, similar dose-dependent

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distribution behavior of Pb2+ was observed (Figure S-17). The consistence results from

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different cell types denotes the wide applicability of the observed cellular distribution

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pattern of Pb2+. In cells, metal ions exist either as free ions or bind to biomolecules. For

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Pb2+, although some Pb2+-binding proteins exhibit strong binding ability and would 17

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compete with GSSH-2TPE, their contents are low. Under the Pb2+ concentrations used in

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this work, the fluorescence of GSSH-2TPE can be used as the indicator for monitoring Pb2+

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in cells.

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The cytotoxicity of GSSH-2TPE was examined with cell viability assay (Figure S-18).

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Even after 24-h incubation with different concentrations of GSSH-2TPE, HeLa cells

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remained >95% viability. Such high biocompatibility makes GSSH-2TPE promising for

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long-term bioanalysis.

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Conclusion

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To conclude, this work presents a biomimetic peptide-based fluorescent sensor GSSH-

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2TPE for tracking Pb2+ in living biosystems. Hard-soft acid theory was successfully

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employed to guide the molecular design and tune the affinity and selectivity of the targeting

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performance. The integration of peptide recognition and AIE effect provides desirable

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sensing features, including specific turn-on response to Pb2+ over 18 different metal ions,

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rapid binding and signal output, as well as high sensitivity with detection limit as low as

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1.5 nM. The ability to image Pb2+ in living cells was demonstrated with high

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biocompatibility, minimal interference from endogeneous biothiols, and no background

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fluorescence. By tracing the Pb2+ in living cells, GSSH-2TPE can monitor changes in Pb2+

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levels, biodistribution thus predict possible damage pathways. Future efforts will exert to

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expand the conjugation chemistry of GSSH to switch the emission wavelength to red or

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infrared regions with the aim to imaging Pb2+ in vivo. Metal-peptide interactions offer

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plentiful tailorable platforms for design bioanalytical and biomedical tools.

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ASSOCIATED CONTENT

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Supporting Information

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Materials and apparatus. HPLC, MS and NMR Characterization, optimization of the

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selectivity of GSSH-2TPE, binding kinetics in solution and in cell, estimation of the

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detection limit and binding affinity, Job’s plot, competitive binding with GSSH, effect of

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biothiols, effect of Ca2+, Mg2+ and Al3+, cell imaging sensitivity of GSSH-2TPE, co-

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staining assays with membrane tracker, imaging Pb2+ biodistribution in different cell lines,

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and cytotoxicity evaluation of GSSH-2TPE. The Supporting Information is available free

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of charge on the ACS Publications website.

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ACKNOWLEDGMENT

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This work is supported by grants from National Natural Science Foundation of China

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(21874141, 21675161, 21621062, and 21705154), Ministry of Science and Technology of

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China (2015CB856303), Chinese Academy of Sciences, and Youth Innovation Promotion

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Association CAS (No. 2015027).

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