Biomimetic Sensing System for Tracing Pb2+ Distribution in

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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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ACS Applied Materials & Interfaces

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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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ACS Paragon Plus Environment

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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

99

coupling

agent,

respectively.

After

peptide

elongation,

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

100

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

101

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

105

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+,

113

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

115

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

119

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

122

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

125

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

128

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

130

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

166

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).

171

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

172

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

173

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

174

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

176

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

177

Information).

178

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

188

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

189

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

191

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

192

(Figure 1).

193

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

194

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-

196

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

197

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

198

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+,

200

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

202

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

204

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

206

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

207

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

208

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

209

(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.

213

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

214

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

215

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

216

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

217

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

218

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

220

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

221

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

225

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

227

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+

229

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

235

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.

238

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-

240

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

241

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

242

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

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rapid and real-time analysis.

244

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

245

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

246

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

247

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

248

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

249

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

250

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

251

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

252

of the peptide sensor.

253

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

254

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

255

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

256

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

257

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

258

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



259

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

260

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

261

Investigation of Pb2+-peptide assembly mechanism.

262

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

263

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

264

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

265

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

266

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

267

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

268

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

269

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

270

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

271

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

272

fluorescence.

273 12


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

275

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

276

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

277

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

278

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

279

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

280

for the formation of coordination complex and supramolecular assembling of fluorescent

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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

292

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

293

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

294

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

295

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

296

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

297

insights into the formation of coordination complex and supramolecular assembly.

298

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

299

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



300

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

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

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

316

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

317

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

318

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

319

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

320

Imaging detection of Pb2+ in living cells.

321

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

322

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

323

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

324

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

325

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


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326

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

327

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

328

sensing Pb2+ in cells.

329

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

330

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

331

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

332

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

333

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

334

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

335

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

336

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

337 338

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

339

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

340

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

341

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

342

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

343

different GSSH-2TPE incubation time.

15



344

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

345

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

346

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

347

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

348

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

349

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

350

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

351

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

352

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

353

Pb2+ treatment remained dark under the same conditions

354

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

355

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

356

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

357

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

358

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

359

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

360

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

361

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

362

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

363

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

364

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

365

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

366

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

367

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

368

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

369

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


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370

pattern also changed. Cytoplasm became strongly emissive with unevenly distributed

371

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

372

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

373

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

374

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

375

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

376

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

377

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

378

destroy biological systems.

379 380

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

381

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

382

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

383

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

384

analysis.

385

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

386

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

387

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

388

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

389

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

390

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

391

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



392

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

393

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

394

in cells.

395

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

396

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

397

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

398

long-term bioanalysis.

399

Conclusion

400

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

401

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

402

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

403

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

404

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

405

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

406

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

407

biocompatibility, minimal interference from endogeneous biothiols, and no background

408

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

409

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

410

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

411

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

412

plentiful tailorable platforms for design bioanalytical and biomedical tools.

413 414

ASSOCIATED CONTENT

415

Supporting Information

18


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416

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

417

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

418

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

419

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

420

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

421

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

422

of charge on the ACS Publications website.

423 424

ACKNOWLEDGMENT

425

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

426

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

427

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

428

Association CAS (No. 2015027).

429 430

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Biomimetic Sensing System for Tracing Pb2+ Distribution in

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