Inorganic Hybrid Materials

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Luminescent Lanthanide-Based Organic/Inorganic Hybrid Materials for Discrimination of Glutathione in Solution and Within Hydrogels Xi Chen, Yuru Wang, Ran Chai, Yang Xu, Huanrong Li, and Binyuan Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02679 • Publication Date (Web): 28 Mar 2017 Downloaded from http://pubs.acs.org on March 30, 2017

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ACS Applied Materials & Interfaces

Luminescent

Lanthanide-Based

Organic/Inorganic

Hybrid

Materials for Discrimination of Glutathione in Solution and Within Hydrogels Xi Chen, Yuru Wang, Ran Chai, Yang Xu*, Huanrong Li*, Binyuan Liu

School of Chemical Engineering and Technology, Hebei University of Technology, Guangrong Dao No.8, Hongqiao District, Tianjin 300130, China. E-mail: [email protected]; [email protected]

Keywords: Nanoclay; Detection; Discrimination; Hydrogel; Glutathione

Abstract: Glutathione (GSH) as a biothiol is an essential peptide related to various diseases. Although multiple strategies for biothiols detection have been developed, there is increasing demand for sensors that can differentiate GSH from cysteine (Cys) and homocysteine (Hcy), owing to the similar structures and thiol groups in these amino

acids.

Herein,

we

report a

Eu3+/LAPONITE®

novel

(Lap)-based

organic/inorganic hybrid material for selective detection of GSH via an “off-on” process. The fluorescence of Eu(DPA)3@Lap-Tris can be quenched by Cu2+ through photo-induced

electron

transfer

Eu(DPA)3@Lap-Tris/Cu2+

system

(PET).

The

induces

addition

the

of

removal

GSH of

into

Cu2+

the from

Eu(DPA)3@Lap-Tris and blocks PET, resulting in the recovery of fluorescence. This proposed assay demonstrates higher selectivity toward GSH than Cys and Hcy, and 1 ACS Paragon Plus Environment


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showed a detection limit of 162 nM within a linear range of 0.5–30 µM. Unlike other GSH selective sensors, this platform could be formed into a hydrogel while its sensitivity was maintaining. The sensitive response to GSH in serum samples makes this platform as an efficient tool for biological applications because of its ease of preparation, high selectivity, good biocompatibility and low toxicity.

INTRODUCTION Glutathione (reduced form, GSH) is a well-known endogenous antioxidant composed with glutamic acid, cysteine (Cys), and glycine. It plays an significant role in various biological processes including Alzheimer’s disease and cardiovascular disease.1 Many kinds of detection assays have been used for the quantification of GSH,

utilizing

high

performance

liquid

chromatography

(HPLC),

2-3

electrochemistry,4 colorimetry,5 and fluorescence.6 Nevertheless, there is still challenging to selectively detect GSH over Cys and homocysteine (Hcy) due to their similar thiol groups and structure, which can interfere with GSH detection.7 Recently, several assays have been proposed for discrimination of GSH from Cys/Hcy. Hu et al. prepared a fluorescent hybrid probe with cyanine and 7-nitrobenzofurazan that responds to GSH and Cys/Hcy with distinctly-separated emissions.6 Jiang et al. reported a tetramethylbenzidine-based probe that responds to GSH over Cys due to inner-filter effects.8 Huang et al. designed a carbon quantum dots-based sensor for discrimination of GSH in two channels.1

However, molecules and tiny

nanomaterials remain challenging to detect due to low biocompatibility, and fast metabolism speed in vivo. Therefore, it is in great demand to find a biological affinity 2 ACS Paragon Plus Environment

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material to design a simple, sensitive assay for selectively detecting GSH over Cys and Hcy. Nanoclays, such as LAPONITE® (Lap) have recently been used as useful inorganic matrix materials owing to their promotion in aqueous systems.9-11 Such dispersions feature low costs, small sizes, easy surface modification, low toxicity, favorable ion-exchange properties and excellent long-term stability. Nanoclays are also a rising star in the field of fluorescent materials, and can promote the usage of rare earth ions in aqueous solution as their adherence to the clay surface reduces water coordination, thus enabling dramatically improved emission intensity.12-13 Nanoclays have been proposed as sensors for detecting various targets, such as metal ions, water, basic molecules, and proteins.14-18 For example, Dai et al. proposed an ion liquid-clay composite to detect myoglobin in electrochemistry method.18 As nanoclays have larger sizes than molecular-based sensors, they have long-term metabolism in vivo for facilitating detection and observation. The novel nanoclay-based sensors will have a great potential for use in future biological applications. Herein, we report a novel Eu3+/ LAPONITE® (Lap)-based organic/inorganic hybrid material for the selective detection of GSH via an “off-on” process, both in solution and within hydrogels. The fluorescence of Eu(DPA)3@Lap-Tris can be efficiently quenched by Cu ions (denoted Cu2+) via photo-induced electron transfer (PET). The addition of GSH into the Eu(DPA)3@Lap-Tris/Cu2+ system induces the removal of Cu2+ from Eu(DPA)3@Lap-Tris and blocks the PET process, resulting in fluorescence recovery. This proposed assay demonstrates high selectivity toward GSH, 3 ACS Paragon Plus Environment


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and shows a detection limit of 162 nM within a linear range of 0.5–30 µM. Unlike other GSH selective sensors, this platform can be formed into hydrogel without losing any sensitivity. To the best of our knowledge, a luminescent nanoclay for GSH detection both in solution and within hydrogel has not yet been reported, and this platform could be used as a case for further GSH detection. The sensitive response to GSH in serum samples makes this platform an efficient tool for biological applications because of its ease of preparation, high selectivity, good biocompatibility and low toxicity. EXPERIMENT SECTION Chemicals EuCl3·6H2O, L-Glutathione (L-GSH, reduced form), L-cysteine (L-Cys), DL-homocysteine

(DL-Hcy),

Tris(Hydroxymethyl)aminomethane

(Tris)

and

Pyridine-2, 6-dicarboxylic acid (DPA, 99%) were bought from J&K Scientific Ltd.(Beijing, China). Metal ions solutions of the perchlorate salts (Al3+, Ca2+, Cd2+, Cr3+, Cs+, Hg2+, Fe2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+, Fe3+, Ag+) and the sodium salt of anions (Ac-, HPO42-, H2PO4-, Cl-, PO43-, CO32-, SO42-, Br-, I-, NO3-, SCN-, HCO3-, HSO4-, SO42-), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophane (Trp), methionine (Met), proline (Pro), glycine (Gly), serine (Ser), threonine (Thr), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), histidine (His), lysine (Lys), arginine (Arg), aspartic acid (Asp), and glutamic acid (Glu) were obtained from Tianjin chemical reagent Co., Tianjin, China. All of the solvents and reagents were used without any further purification. 4 ACS Paragon Plus Environment

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Instruments The UV−Vis absorption spectra were tested with an UV−visible Agilent cray 100 spectrometer with a quartz cuvette. Infrared (IR) spectra were obtained with a Bruker Vector 22 spectrophotometer by using KBr pellets for solid samples from 400-4000 cm-1 at a resolution of 4 cm-1. The Eu content was measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES), IRIS advantage, Thermo, U.S.A. The fluorescence spectra were performed with a near-infrared spectrometer (FS920P, Edinburgh Instruments) with a 450 W xenon lamp as the steady-state excitation source, a double excitation monochromator (1800 lines·mm-1), an emission monochromator (600 lines·mm-1), a semiconductor cooled Hamamatsu RMP928 photomultiplier tube, and an integrating sphere for absolute quantum yield measurements. Samples for thermogravimetric (TGA) studies were transferred to open platinum crucibles and analyzed by using a SDT-TGQ 600, at a heating rate of 10 °C min-1 using dried air as purging gas. Transmission electron microscopy (TEM) images chemical mapping were recorded with a Tecnai G2 F20, (FEI Co. U.S.A.) operated at an accelerating voltage of 200 kV. Absorbance data for the GSH determination was measured at 412 nm by a SpectraMax i3 Multimode microplate detection platform (Molecular Devices, USA). The hydrodynamic size and ζ-potentials were tested at 25 °C and recorded with a Zetasizer Nano ZS (Malvern, British) with a 633 nm He−Ne laser. Synthesis of Eu(DPA)3@Lap-Tris

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As shown in scheme 1, Lap (1.0 g) was dispersed in distilled water (20 mL) under sonication (BRANSON 2510, 100 W) for 30 min to obtain homogeneous solutions. EuCl3 (0.1 M, 3 mL) were added to solution with stirring at 90°C for 24 h to replace for Na+ in ion exchange

19

. The product was collected by centrifugation and washed

with ultrapure water several times. Then, the precipitate was dissolved in water to form a milky solution. DPA (0.1 mmol) was added into the distilled water. In order to improve the solubility of carboxylic acid, a moderate amount of NaHCO3 was dropped in the mixture 20. When the solution became clearly, the PH was adjusted to be neutral by adding Tris(Hydroxymethyl)aminomethane (Tris), then the mixture stirred at 90°C for 24 h 21. The resulting product, named as Eu(DPA)3@Lap-Tris was filtered out and washed with ultrapure water, followed by air-drying. Detection of Cu2+ and GSH For detection of Cu2+, different concentrations of Cu2+ were mixed with 5 mg/mL Eu(DPA)3@Lap-Tris aqueous solution. (The concentration of Cu2+ was 0, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 30, 40 and 50 µM). UV–vis and fluorescence spectra were measured after 10 min 22. For detection of GSH, the test solutions were prepared by the Eu(DPA)3@Lap-Tris 5 mg/mL then added the copper nitrate solution and ensure the concentration of Cu2+ was 10 µM. The solutions of GSH were directly added in it (the concentration of GSH was 0, 0.5, 0.8, 1, 2, 5, 8, 10, 20, 30, 40 and 50 µM). UV–vis and fluorescence spectra were taken after 10 min. To evaluate the selectivity of the Eu(DPA)3@Lap-Tris, Metal ions solutions of the 6 ACS Paragon Plus Environment

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perchlorate salts (Al3+, Ca2+, Cd2+, Cr3+, Cs+, Hg2+, Fe2+, K+, Li+, Mg2+, Mn2+, Na+, Ni2+, Pb2+, Zn2+, Fe3+, Ag+) or the sodium salt of anions (Ac-, HPO42-, H2PO4-, Cl-, PO43-, CO32-, SO42-, Br-, I-, NO3-, SCN-, HCO3-, HSO4-, SO42-) were prepared with double distilled water, respectively. All samples were constituted by interferents 20 µL (1 mM), Eu(DPA)3@Lap solution 1480 µL (5 mg/mL) and ultrapure water (0.5 mL) at room temperature and were shaken for 1 min. While the selective detection of the Eu(DPA)3@Lap-Tris-Cu(II) to amino acid was measured in the same process, (Ala, Arg, Asp, Glu, Gly, His, Ile, Tyr, Leu, Met, Phe, Pro, Ser, Thr, Trp, Val, Gln, Asn, Cys) were prepared in distilled water. All samples were constituted by interferents 40 µL (1 mM), Eu(DPA)3@Lap-Tris-Cu(II) solution 1460 µL and ultrapure water (0.5 mL) at room temperature and were shaken for 1 min. Detection of GSH in real samples. The detection of GSH in fetal bovine serum was carried out by using Eu(DPA)3@Lap-Tris. A commercial GSH assay kit (Beyotime Biotechnology, Jiangsu, China) based on 5,5-dithio-bis(2-nitrobenzoic)acid (DTNB) method was performed as a reference. Absorbance was measured at 412 nm by a SpectraMax i3 Multimode microplate detection platform. Preparation of the hydrogels and transparent film A 5 mg/mL solution of Eu(DPA)3@Lap-Tris or Eu(DPA)3@Lap-Tris with different concentration of Cu2+ and GSH in water was added in centrifuge tube (2 mL), with evapo-ration of water at 50 oC in air to form a transparent gel. 7 ACS Paragon Plus Environment


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Eu(DPA)3@Lap (0.05 g) was dropped in 10 mL water then an aqueous solution of PVA (0.02 g/mL) was addedobtain a clear solution. The mixture was stirred at room temperature for 1 h. A film was formed by dropping the solution on micro slide glasses, followed by evapo-ration of water at 50 oC in air. Cell Viability by MTT Prior to detection in the biological environment the long-term cytotoxicity of probe to the HeLa cells (human cervical adenocarcinoma cell line) was evaluated using the MTT assay method. All cells harvested the cell density was adjusted to 5×104 cells per well and allowed to grow over 24 h at 37 °C. After the medium was replaced with 90 µL fresh Dulbecco modified Eagle’s medium (DMEM), then different concentrations of determine and suspensions (5, 10, 50, 100, 250 and 500 µg mL−1) were added. After that, the cells were cultivated for 24 h, and 20 µL of 5 mg/mL MTT solution was added to each cell well. After incubation for 4 h, the culture medium was discarded, and 150 µL of dimethyl sulfoxide was added. The obtained mixture was shaken for 15 min in the dark at room temperature. The absorbance of each well was measured at 490 nm for the calculation of cell viability. At least three independent experiments were performed under identical conditions, its optical density was measured by using a microplate reader (Thermo) 23. RESULTS AND DISCUSSION


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Scheme 1. Schematic representation of Eu(DPA)3@Lap-Tris preparation and GSH detection.

Synthesis and Characterization of Eu(DPA)3@Lap-Tris Nanohybrids LAPONITE® (Lap) nanodisks possess a unique two-dimensional layered structure with six octahedral magnesium ions between two layers of four tetrahedral silicon atoms. The unique structural characteristics of Lap make it possible to efficiently encapsulate molecules within the Lap interlayer space.24 Recently, various lanthanide-doped nanoclays have been prepared using Lap as a platform for fluorescence applications.25 In this work, Eu(DPA)3@Lap-Tris was prepared by facile reflux according to a previously-reported method with minor modifications.26 This hybrid material showed good solubility because of the hydroxyl groups on its interface.27 We found that the fluorescence of Eu(DPA)3@Lap-Tris can be quenched by Cu2+, and recovered after the addition of GSH, demonstrating an “off-on” process with regards to GSH detection. Furthermore, this hybrid material can exist as a hydrogel, while maintaining its detection ability for GSH. The preparation and GSH detection of Eu(DPA)3@Lap-Tris are illustrated in Scheme 1. 9 ACS Paragon Plus Environment


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Figure 1. (A) TEM images and the size distribution of Eu(DPA)n@Lap-Tris obtained from (B) TEM and (C) DLS. (D) FTIR spectra of Lap, DPA, Tris, Eu(DPA)3@Lap and Eu(DPA)3@Lap-Tris. (E) TG curves of Lap, DPA, Tris, Na3[Eu(DPA)3], Eu(DPA)3@Lap-Tris.

As shown in Figure 1A and 1B, transmission electron microscopy (TEM) indicated that the hybrid material was well-dispersed in aqueous solution, with an average feature diameter of 42 nm, similar to Lap nanodisks (~40 nm).

26

Dynamic light

scattering (DLS) measurements revealed that the size of Eu(DPA)3@Lap-Tris ranged from 30–60 nm (Figure 1C), which was consistent with TEM measurements. Fourier transfer infrared (FTIR) experiments were conducted to discern the formation of Eu(DPA)3@Lap-Tris. The FTIR spectra of Lap, DPA, Tris, Eu(DPA)3@Lap and Eu(DPA)3@Lap-Tris are shown in Figure 1D. The peaks at 1007 cm–1 and 3436 cm–1 in the Lap and Eu(DPA)3@Lap spectra are attributed to the −Si−O− stretching vibration and −OH bending vibration in the Lap nanodisks, respectively. Compared to DPA, the COOH group stretching vibration at 1700 cm–1 is absent and a new band appears at approximately 1633 cm–1 in Eu(DPA)3@Lap due to 10 ACS Paragon Plus Environment

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the nitrogen of pyridine that participates in the coordination of Eu.28 The shift of the OH stretching vibration in Eu(DPA)3@Lap-Tris to 3351 cm–1 suggests that the hydroxy groups of Tris are involved with the Lap platelets. Moreover, the appearance of a peak in the Tris and Eu(DPA)3@Lap-Tris spectra at 1586 cm–1 was ascribed to – NH2 deformation vibrations.29 The thermal stabilities of the nanoclays, DPA, and Tris were investigated by thermogravimetric analysis (TGA). The TGA curves of the samples prepared under the same conditions are shown in Figure 1E. The TGA traces indicated two major steps during the decomposition of the material. The first change occurrs at approximately 180 °C, attributed to the evaporation of water and decomposition of functional groups. The more prominent change appears at approximately 230 °C due to the decomposition of the organic compounds (DPA, Tris). The final residual yield was 63.72 wt%. These results indicate that Eu(DPA)3@Lap-Tris possesses good thermal stability, and Tris was attached to the clay platelets surface.30 These results reveal that Eu(DPA)3@Lap-Tris features three functional units: Eu(DPA)3 as the fluorescence source, Lap as the matrix, and Tris as the modification group to improve properties of the hybrid material. Optical Properties UV-vis absorption spectra of the ligands and the Eu3+ hybrid materials were recorded between 240 and 320 nm in solution as depicted in Figure 2A. Inspection of the absorption behavior of DPA revealed the presence of a peak situated at 270 nm. This coincides with the π– π* absorption observed for the corresponding complexes, 11 ACS Paragon Plus Environment


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which mainly involve orbitals located on the pyridine ring and carbonyl functional groups. Furthermore, the absorption maxima peaks of the hybrid materials underwent a slight red shift at 280 nm, due to the formation of larger conjugated rings when the ligands are coordinated to the Eu3+ ions. 31 There was no apparent absorption for Lap, indicating that the europium complexes were loaded as expected. The excitation spectra of the nanohybrid solutions exhibited a similar excitation band with a maximum peak at 282 nm (Figure 2B). Figure 2C show emission spectra of Eu(DPA)3@Lap-Tris in solution, with characteristic 5D0→7FJ transitions at 590 and 615 nm for J = 1 and 2, respectively. By contrast, the 5D0→7F2 transition is the result of induced electric dipole character and its corresponding intensity at 615 nm is very sensitive to the coordination environment in question, yielding a red emission color in this instance. 32 Protonation of rare earth complexes can impact the coordination to Eu3+, and this coordination affects the luminescence intensity. The proposed mechanisms can be further supported by the following observations regarding the environment of Lap. In this work, we designed the protonation of the LAPONITE® platelets and inspected the influence of protonation between Eu(DPA)3@Lap and Eu(DPA)3@Lap-Tris (Figure 2D).26 Following this reasoning, we found that the remarkable influence of Tris on the luminescence intensity of Lap arises from the Tris buffered the proton activity outside of the Lap channels.33 Each buffer molecule attached to the channel exchanges the cations that control the proton strength. This is supported by the observation in Figure 2D, where a very large intensity change occurs at low pH in the 12 ACS Paragon Plus Environment

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case of Eu(DPA)3@Lap. Therefore, Eu(DPA)3@Lap-Tris is suitable as a platform for the subsequent experiments.

Figure 2. (A) UV spectra of Lap, DPA, and Eu(DPA)3@Lap-Tris. (B) Excitation and (C) emission fluorescence spectra of Eu(DPA)3@Lap (black lines) and Eu(DPA)3@Lap-Tris (red lines) at the same concentration in aqueous solution. (D) Fluorescence intensity of Eu(DPA)3@Lap (red) and Eu(DPA)3@Lap-Tris (black) at various pH values.

Sensing and Selectivity of Cu2+ For

further

studies,

we

found

that

the

fluorescence

intensity

of

Eu(DPA)3@Lap-Tris was decreased upon the addition of Cu2+ (using Cu(NO3)2 as the copper source). Thus, we studied the relationship between the nanohybrids and Cu2+.

In the UV-vis absorption spectra (Figure S1A), the intensity of two absorption peaks of Eu(DPA)3@Lap-Tris at 272 nm and 280 nm were enhanced with the copper nitrate concentrations increased, meanwhile a significant blue shift was observed. It 13 ACS Paragon Plus Environment


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implied that the carboxyl in DPA and nitrogen atoms in Tris might coordinate Cu2+ to form a chelate and the environment around the Eu3+ ions was disturbed to initiate a PET process. The UV–vis absorption of Eu(DPA)3@Lap-Tris with other metal ions and anions was further examined in aqueous conditions. As shown in Figure S1B and S1C, the maximum absorption wavelength of Eu(DPA)3@Lap-Tris is approximately at 272 nm. Upon addition of different interfering substances, a blue shift of the absorption from 272 to 268 nm was only observed in the presence of Cu2+. Moreover, the coordination was further confirmed by the less negative ζ-potential of Eu(DPA)3@Lap-Tris (–10.1 mV) compared to that of Eu(DPA)3@Lap (–35.2 mV). It demonstrated that Tris could enhance the zeta potential of nanoclay to avoid the electrostatic adsorption between nanoclay and Cu2+, and participate in the coordination between nanoclay and Cu2+. Figure 2B showed that pH can influence rare-earth complexes and transition metal complexes in the coordination of organic compounds, leading to differences in fluorescence intensity. In order to obtain an optimal pH value for developing a sensitive fluorescence sensor for Cu2+, the fluorescence of Eu(DPA)3@Lap-Tris-Cu in solutions of different pH values (pH = 4–9) were investigated in this work. As shown in Figure S1D, the fluorescent quenching intensity displayed a decreasing trend after an initial ascent, and the maximum fluorescence quenching appears at a pH of 8. According to the literature, the difference in growth rates between in the presence and absence Cu2+ should arise from the different reaction activities of the sensors, which may be attributed to the different coordination fashions of Cu2+ with the sensors at 14 ACS Paragon Plus Environment

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different pH values. Under weak alkaline conditions, the deprotonation of the rare-earth ion in Lap is favored, which enhances the change between cases with and without Cu2+. However, when the pH is higher than 8, the fluorescence quenching intensity gradually declines. It suggests that the coordination in the resulting compound is destroyed due to the hydroxide ions in the phosphate buffer solution. Meanwhile, a high pH also causes a Cu(OH)2 precipitate to form. Therefore, the pH value of the sensor solution is approximately 8 in this work, which affords the best sensitivity and stability for Cu2+ detection.34

Figure 3. (A) Fluorescence response of Eu(DPA)3@Lap-Tris at varying Cu2+ concentrations (0– 50 µM). (B) The calibration curve of ∆F versus Cu2+ ions from 0.5 µM to 10 µM. (C) Fluorescence images of Eu(DPA)3@Lap-Tris hydrogel upon 365 nm excitation. Fluorescence responses of Eu(DPA)3@Lap-Tris in the presence of (D) metal ions and (E) anions. All interfering substances were added in water at a concentration of 10 µM. F0 and F1 are the fluorescence intensity in the absence and presence of ions, respectively.

As shown in the inset of Figure 3A, the luminescence of as-prepared 15 ACS Paragon Plus Environment


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Eu(DPA)3@Lap-Tris is quenched in the presence of Cu2+, with the emission spectra displaying a gradual decrease in emission intensity with increasing Cu2+ concentration (0 to 50 µM). A linear relationship was observed between the fluorescence intensity of Eu(DPA)3@Lap-Tris and the concentration of Cu2+ ions over a range of 0.5 to 10

µM (Figure 3B). The limit of detection (LOD) is estimated to be 92 nM. Eu(DPA)3@Lap was also used as a platform for Cu2+ detection. We found that Eu(DPA)3@Lap-Tris exhibited a higher detectability for Cu2+ detection than Eu(DPA)3@Lap group (Figure S1E). It indicated that the introduction of Tris can improve the sensitivity of the lanthanide-based nanoclay. Notably, this system could be used in a hydrogel form, while retaining its previously-observed fluorescence quenching ability (Figure 3C). To assess the selectivity of the Eu(DPA)3@Lap-Tris platform for Cu2+, a number of different interfering species were added in solution to investigate their impact on this platform. Figure 3D revealed that Eu(DPA)3@Lap-Tris has excellent selectivity for Cu2+ over other metal cations and anions. In addition, 10 µM Ag+ had a minimal effect on the fluorescence intensity of Eu(DPA)3@Lap-Tris. Few emission intensity changes were observed in the presence of these interfering metal ions, demonstrating that this assay method was highly selective for Cu2+ and was unaffected by the presence of other ionic species.

35

The emission response in the presence of anions

was examined under the same conditions, as anions present in solution can be another factor that affects such sensing systems (Figure 3E). No substantial changes were observed from the anions tested, demonstrating that anion interference in solution is a 16 ACS Paragon Plus Environment

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minimal factor on this system. Establishment of a Sensing System for GSH

Figure 4. (A) The effect of Cu2+ concentration on the fluorescence recovery efficiency of fluorescence turn-on probe. (B) The relationship between the fluorescence intensity of 10 µM Eu(DPA)3@Lap-Tris-Cu2+ and the incubation time with the addition of 1, 5, 10, 20 µM GSH.

For the kinetic study of fluorescence recovery effects, the influence of incubation time was studied and the results are shown in Figure 4A. The fluorescence intensity of Eu(DPA)3@Lap-Tris-Cu2+ increased immediately after adding GSH and the recovered fluorescence intensity remained unchanged with the increasing incubation time.

This

result

explains

that

the

fluorescence

recovery

processes

of

Eu(DPA)3@Lap-Tris-Cu2+ with GSH finished in a short time and the recovery processes is stable with the increasing incubation time. Fluorescence probes with Cu2+ could be used as sensors for GSH detection, because GSH can recover its fluorescence by coordinating with Cu2+ as shown in previous works.

1, 6

To optimize

the conditions for GSH detection, Eu(DPA)3@Lap-Tris was first prepared with 10 µM GSH. Then, 0.5–20 µM Cu2+ solutions were added to different samples and the fluorescence intensities of the hybrid platform with various concentrations of Cu2+ 17 ACS Paragon Plus Environment


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were monitored.36 Significant changes in the fluorescence intensity were observed as the concentration of Cu2+ were varied (Figure 4B). When the concentration of Cu2+ was 10 µM, the fluorescence intensity underwent its most drastic change. Further, a series concentrations (1.0, 2.0, and 3.0 times) of GSH were added into a certain concentration of Cu2+. Figure S2 showed that the fluorescence intensity of 10 µM Cu2+ group exhibited the most enhancements. Based on Figure 4B and Figure S2, the determination of different concentration of GSH is most obvious for 10 µM Cu2+. We therefore used Eu(DPA)3@Lap-Tris-Cu2+ (10 µM) as the test condition for further studies.

Sensing and Selectivity of the System to GSH. We also investigated the effects of GSH on the UV–visible absorption spectra of this system to further understand its behavior in aqueous systems (Figure S3A). As expected, adding increased concentrations of GSH to Eu(DPA)3@Lap-Tris-Cu2+ (10 µM) resulted in a gradual decrease of the absorption peaks at 270 nm and 280 nm. To further understand the high selectivity of the probe for GSH over other biological thiols, we

examined

the

changes

of

UV–visible

absorption

spectra

of

Eu(DPA)3@Lap-Tris-Cu2+ (10 µM) after treatment with other amino acids (Figure S3B). No obvious changes were observed in the UV–vis absorption spectra of the probe ensemble in the presence of the test amino acids.37 These results revealed that the probe had good selectivity for GSH detection compared to the other test amino acids, including Cys and Hcy.38


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Figure 5. (A) Fluorescence spectra of Eu(DPA)3@Lap–Tris–Cu2+ in the presence of different GSH concentrations. (B) Calibration curve of ∆F for various GSH concentrations (0–30 µM) in Eu(DPA)3@Lap–Tris–Cu2+. Fluorescence images (λex=254 nm) of Eu(DPA)3@Lap–Tris–Cu2+ in the state of (C) hydrogel and (D) film after addition of different amino acids (10 µM). (E) Fluorescence spectra of Eu(DPA)3@Lap–Tris–Cu2+ and Eu(DPA)3@Lap–Tris–Cu2+ with GSH (30 µM) or EDTA (30 µM) in solution. (F) Fluorescent responses of Eu(DPA)3@Lap–Tris–Cu2+ (10 µM) in aqueous solution with different amino acids. F0 and F1 denoted the fluorescence intensity in the absence and presence of amino acids (30 µM), respectively.

Notably, only GSH induced a “turn on” response as shown in Figure 5A, where the 19 ACS Paragon Plus Environment


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fluorescence emission at 615 nm increased gradually as the concentration of GSH was increased due to the formation of free Eu(DPA)3@Lap-Tris upon addition of GSH to Eu(DPA)3@Lap-Tris-Cu2+. The detection limit for GSH was calculated to be 162 nM, and there is also excellent linearity in the concentration range of 0.5−30 µM (Figure 5B). These nanohybrids have similar detectability as shown in the previous works (Table S1). Interestingly, Eu(DPA)3@Lap-Tris-Cu2+ can also form a hydrogel, while maintaining the recovery of fluorescence with GSH (Figure 5C). An apparent enhancement of the fluorescence due to GSH is appeared, which was not observed with other amino acids in transparent hydrogel (Figure 5D). All of these results demonstrated that this hybrid material has great potential for GSH detection in solution and in hydrogels. Compared to other detection materials, soft-gel materials offer the potential for more sensitive stimuli-responsive properties due to their reversible covalent chemistry and supramolecular interactions. We therefore specifically focused on characterizing the stimuli-responsive properties of the Lap-gel. As expected, gel−sol phase transitions occurred when the water evaporated at a particular rate. In Figure S3, an obvious change to the fluorescence process was observable with the naked eye. It is obvious that the fluorescent intensities decreased dramatically by increasing the concentration of Cu2+, and the fluorescence intensity increased as the concentration of GSH increased. Surender et al. proposed a lanthanide cyclen-based enzymatic assay for urinary tract infections within hydrogels.39 The hydrogel has a better potential in biological applications than in solution. In a similar light, our hydrogel sensor based 20 ACS Paragon Plus Environment

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on hybrid materials has a broad possible space for development, especially in biological systems. The “turn on” fluorescence response of Eu(DPA)3@Lap-Tris-Cu2+ towards GSH was further tested through the addition of EDTA (Figure 5E). Upon addition of increasing

the

amounts

of

GSH,

the

fluorescence

intensity

of

the

Eu(DPA)3@Lap-Tris-Cu2+ solution increased gradually, and the emission intensity at 615 nm reached a maximum at a GSH concentration of 30 µM. Similar fluorescence recovery results were observed with an EDTA concentration of 30 µM (Figure 5E). This demonstrated that the fluorescence change is caused by the PET process, where the formation of a complex between Eu(DPA)3@Lap-Tris and Cu2+ induces the energy transfer. Upon addition of GSH, Cu2+ forms a complex with GSH to block the PET process, releasing Eu(DPA)3@Lap-Tris for the recovery of fluorescence emission. Figure 5F shows a comparison of experimental data for various amino acids. An apparent fluorescence enhancement was observed for Eu(DPA)3@Lap-Tris-Cu2+ after addition of 30 µM of GSH. In contrast, a minor fluorescence enhancement was appeared after addition of various amino acids such as Ala, Arg, Asp, Glu, Gly, His, Ile, Tyr, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Val, Gln, Asn (30 µM). Furthermore, the addition of Cys and Hcy to the Eu(DPA)3@Lap-Tris-Cu2+ solution led to a small enhancement in the fluorescence intensity. This demonstrates that our nanohybrids have excellent selectivity, and an effective power to discriminate GSH over Cys and Hcy. 21 ACS Paragon Plus Environment


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Biocompatibility and Application of Nanohybrids Figure S5 presents cell viability data of HeLa cells incubated with different concentrations of Na3[Eu(DPA)3], Eu(DPA)3@Lap, and Eu(DPA)3@Lap-Tris for 24 h. HeLa cell viabilities remained at approximately 78% even at a high concentration of 500 µM for 24 h in Eu(DPA)3@Lap-Tris, indicating that Tris can efficiently improve the biocompatibility of these nanohybrids. These results demonstrated that Eu(DPA)3@Lap-Tris has low cytotoxicity at both low and high concentrations, and has great potential in biological applications. The stability of the probe is also an important factor affecting its future applications. Figure S6A demonstrates that the probe can be stored for long periods of time at room temperature under ambient conditions with minimal changes to its luminescence properties. It is also stable in water or air over long periods of time, which can be utilized in its detection applications. In order to study the effect of temperature on the detection capabilities, additional experiments were carried out at 30 and 90 °C. From Figure S6B, we found that the fluorescence intensity of Eu(DPA)3@Lap-Tris-Cu2+ was unchanged at different temperatures, suggesting that Eu(DPA)3@Lap-Tris-Cu2+ is stable under increasing temperatures. The anti-photobleaching experiment was performed in Figure S7. A slight (

Inorganic Hybrid Materials

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