A long lifetime ratiometrically luminescent tetracycline nanoprobe based on Ir(III) complex-doped and Eu3+-functionalized silicon nanoparticles

Journal of Hazardous Materials 386 (2020) 121929

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A long lifetime ratiometrically luminescent tetracycline nanoprobe based on Ir(III) complex-doped and Eu3+-functionalized silicon nanoparticles

T

Xiaotong Lia,1, Kaimei Fana,1, Ruimei Yangb, Xiuxiu Dua, Baohan Qua, Xiangmin Miaoc,*, Lihua Lua,* a

College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, China College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China c School of Life Science, Jiangsu Normal University, Xuzhou 221116, China b

G R A P H I C A L A B S T R A C T

A R T I C LE I N FO

A B S T R A C T

Editor: R Teresa

Different from fluorescent dyes-doped or carbon materials-based ratiometric tetracycline nanoprobes, herein, a new Ir(III) complex-doped and europium(III) ion (Eu3+)-functionalized silicon nanoparticles (Ir(III)@SiNPsEu3+) with long luminescent lifetimes was firstly fabricated for selective detection of tetracycline (TC) in complex systems through time-resolved emission spectra (TRES) measurement. In the presence of TC, the red phosphorescence of Eu3+ is greatly enhanced by adsorption energy transfer emission (AETE) of TC, while the strong green luminescence of Ir(III)@SiNPs is quenched by the inner filtration effect (IFE) of TC. Based on these striking emission changes, Ir(III)@SiNPs-Eu3+ can sensitively detect TC in the linear range of 0.01–20 μM with a detection limit of 4.9 × 10–3 μM. Benefitting from the long lifetime of Ir(III)@SiNPs-Eu3+, the nanoprobe demonstrates excellent TC detection performance through TRES in high background system of 5 % human serum. Furthermore, the formed Ir(III)@SiNPs-Eu3+/TC complex can be used to sensitively recognize Hg2+ via a ratiometric luminescence mode. Notably, the cytotoxicity of Ir(III)@SiNPs-Eu3+ is very low and thus the sensitive monitoring the detection of Ir(III)@SiNPs-Eu3+ to TC and Hg2+ also works well in porcine renal cells, demonstrating high application potential in real samples.

Keywords: Iridium(III) complex Europium(III) ion Tetracycline Time-resolved emission spectra Luminescence Cell imaging

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Corresponding authors. E-mail addresses: [email protected] (X. Miao), [email protected] (L. Lu). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jhazmat.2019.121929 Received 11 October 2019; Received in revised form 17 December 2019; Accepted 17 December 2019 Available online 18 December 2019 0304-3894/ © 2019 Elsevier B.V. All rights reserved.

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1. Introduction

detection platforms. SiNPs are important nanomaterials and has received extensive attention due to their unique properties of easy modification, large load capacity, good water dispersity, excellent biocompatibility and low cost (Schnappinger and Hillen, 1996; De Plano et al., 2018; Chen et al., 2019). Of note, SiNPs are easily doped with transition metal complexes, whose original luminescence can be preserved. Therefore, it is an ideal carrier for the fabrication of Ir(III) complex based TC nanoprobe. Inspired by these concepts, a Ir(III) complex-doped silicon luminescent nanomaterial Ir(III)@SiNPs was fabricated as a reference to establish a novel long-lived ratiometrically luminescent nanoprobe Ir (III)@SiNPs-Eu3+ for TC detection. Ir(III)@SiNPs have rich carboxyl groups on their surfaces and can be covalently grafted with APTES and Cit in sequence to fabricate coordination pockets for Eu3+. In the presence of TC, its wide UV absorption range covers the excitation spectrum of Ir(III)@SiNPs and leads to the quick decrease in the luminescence intensity of Ir(III)@SiNPs, while the sharp luminescent emission peak of Eu3+ appears concomitantly and the intensity is gradually increased with TC concentration. In this assay, Ir(III) complex not only serves as a luminescent reference signal through the IFE of TC, but also offers a long-lived phosphorescent illuminant which plays pivotal role in TRES testing. In virtue of the long-lived phosphorescence of Ir(III) complex and Eu3+, the fabricated nanoprobe can perform ratiometric assay for the TC in complicated biological systems through TRES technique, which cannot be conducted by the reported strategies based on organic dye or carbon nano-materials. To the best of our knowledge, this is the first Ir(III) complex-based ratiometric TC nanoprobe with high application potential in biological fields.

Tetracycline (TC) is a broad-spectrum antibiotic through inhibiting bacterial growth. Its large-scale use has caused serious problems, especially in livestock industry. Residues of TC have been found in animal-derived products such as milk and meat. Accumulative TC can cause allergic reactions, gastrointestinal disorders and liver toxicity, heavily destroying human health. In addition, bacterial resistance is also a challenge for the development of new antibiotics. To date, a number of detection strategies, such as, electrochemistry (Liu et al., 2016; Taghdisi et al., 2016; Feng et al., 2019; Wang et al., 2019; Zhou et al., 2012; Wong et al., 2015), colorimetric (Wu et al., 2020; He et al., 2013; Ramezani et al., 2015; Qi et al., 2018), Raman (Lee et al., 2015; Zhao et al., 2016; Dhakal et al., 2018) and fluorescence sensors (Wei and Chen, 2019; Shi et al., 2017; Hou et al., 2016; Jalalian et al., 2018; Salinas et al., 2011; Zhang and Chen, 2016; Chen et al., 2017), have been developed to effectively monitor TC. Among these strategies, fluorescent strategies are received increasing attention in biological systems due to their high sensitivity, rapid response and simple operations. Especially, it has recently popped up ratiometric fluorescent measurements, which can eliminate the adverse effects of environmental background and provide accurate measurement via the self-referencing of two emission bands under quantitative conditions (Tu et al., 2016; Fu et al., 2016). TC contains a β-diketone configuration and exhibits an obvious absorption band around 270 nm, so the mostly developed ratiometrically fluorescent TC probes are based on the absorption energy transfer emission (AETE) effect of TC, which transfer excitation energy to Eu3+ through “antenna effect” (Tan and Chen, 2012; Li et al., 2018; Arnaud and Georges, 2001; Xu et al., 2019). In this case, the coordinated water molecules in the detection system quenches the luminescence of Eu3+, which causes the emission intensity of Eu3+/ TC complex is rather weak. When the coordinated water molecules are substituted by a suitable chelating agent such as citric acid (Cit), the emission intensity of Eu3+/TC complex are enhanced, and hence improve the sensitivity and selectivity of TC nanoprobe. In addition, the broad UV absorption of TC can quench the emission of certain luminescent materials by inner filtration effect (IFE) (Lin et al., 2016). In virtue of this phenomenon, two types of ratiometrically fluorescent TC nanoprobes have been developed. One type is that the reference fluorescent signal is unchanged with the increase of Eu3+ luminescence intensity in the presence of TC. For example, Jia and colleagues used palygorskite nanomaterial as stable fluorescent reference to establish a novel ratiometric TC sensing platform Xu et al. (2018), and Liu’s group constructed a ratiometric fluorescent TC probe through employing cyclen functional carbon dots as a fluorescent reference Shen et al. (2018). The other type is that the reference fluorescent signal is dropped down when the signal of Eu3+ is increased in the presence of TC, which is usually more sensitive than the first type ascribed to its lever effect. For instance, Zheng’s group constructed a ratiometric system based on graphene quantum dots, whose fluorescence intensity is reduced in the presence of TC (Li et al., 2018). Qin and his colleagues established a Eu3+ functionalized SiNPs, demonstrating decreased fluorescence intensity upon the addition of TC (Li et al., 2017). Although both of these ratiometrically fluorescent detection systems can perform sensitive and selective TC assay in buffered solution, they cannot match the long lifetime of Eu3+ because the reference signals in these systems usually come from short-lived fluorescent materials. Therefore, this hinders their usage in complicated biological environment, wherestrongly autofluorescent background interferes with the fluorescence of these TC nanoprobes. Using transition metal complex as luminescent reference can overcome this shortcoming. Ir(III) complexes, as a typical phosphorescent molecules, possess large Stokes shift, long life time and adjustable emission wavelength. Thus, they are promising candidates for application in luminescence-based detection (Kim et al., 2017; Park et al., 2019; Liu et al., 2017; Yang et al., 2011; Li et al., 2012). However, Ir(III) complexes are seldom used in ratiometrically luminescent

2. Methods 2.1. Chemicals and materials 3-Aminopropyltriethoxysilane (APTES, 99 %), tetraethoxysilane (TEOS, 98 %), N-hydroxysuccinimide (NHS), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide chloride (EDC), europium chloride (EuCl3·6H2O, 99.99 %) and tris(hydroxymethyl)aminomethane (Tris) were purchased from Bailingwei Technology Co., Ltd., Beijing, China. Tetracycline (TC, 99 %) and citric acid (Cit, 98 %) were purchased from Shanghai Aladdin Co., Ltd., China. All reagents were of analytical grade and were used without further processing. 2.2. Preparation of Ir(III)@SiNPs The synthesis of complex 1 was presented in electronic supporting information. 2.5 mg Ir(III) complex 1 was mixed with 37 mL anhydrous ethanol, 2 mL ultrapure water, 1 mL APTES and 1 mL TEOS. Then, 4 mL ammonia water was added slowly and stirred for 24 h at room temperature. The NH2-functionalized nanoparticles Ir(III)@SiNPs were isolated by centrifugation and washed in turn with anhydrous ethanol and ultrapure water twice for each. The nanoparticles were freeze-dried under vacuum for next use. 2.3. Preparation of Ir(III)@SiNPs-Eu3+ As described in the literature (Xu et al., 2018), EDC and NHS were added to Cit aqueous solution (the mass ratio of EDC:NHS:Cit was 1:1:6). Then the prepared Ir(III)@SiNPs (100 mg per 20 mL of ultrapure water) was added to the above solution, and the mixture was stirred at room temperature for 8 h. Afterwards, the mixture was separated by centrifugation and washed twice with ultrapure water. The purified Citmodified Ir(III)@SiNPs was dispersed in EuCl3·6H2O aqueous solution (3 mL, 10 μM) and stirred at room temperature for 8 h. Finally, the nanoprobe Ir(III)@SiNPs-Eu3+ can be obtained through centrifugation and washed several times with deionized water to remove residual EuCl3. The solid was prepared into a solution with the concentration of 2

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100 mg mL–1 in ultrapure water.

light scattering (DLS) analysis, respectively. As shown in Fig. 1A and 1B, Ir(III)@SiNPs-Eu3+ are highly dispersed nanoparticles, indicating easy dispersibility in aqueous solution. Their size distribution ranges from 80 nm to 160 nm with an average diameter of 120 nm. The wide scan X-ray photoelectron spectroscopy (XPS) of NH2-modified SiNPs and Ir(III)@SiNPs-Eu3+ are collected to investigate their composition and chemical bonds (Fig. 1C). There are four peaks at 103.7, 287.5, 399.5 and 530.2 eV, which are attributed to Si2p, C1s, N1s and O1s, respectively. Meanwhile, the wide scan spectrum of Ir(III)@SiNPs-Eu3+ exhibits additional peaks at 62.6 and 1134.0 eV, which are respectively attributed to the Ir4f and Eu3d in Ir(III)@SiNPs-Eu3+. The high resolution XPS spectra of C1s, N1s, O1s and Si2p (Fig. S1A–D) verify the presences of CeC, CeH, CeN, CeO, SieC, SieO and NeH bonds in NH2-modified SiNPs. The appearance of new peaks in C1s, N1s, O1s and Si2p of Ir(III)@SiNPs-Eu3+ (Fig. S2A–D) further confirm the presence of NeC]O, HOeC]O and O]C bonds. The Zeta potentials of Ir(III)@ SiNPs, NH2-modified Ir(III)@SiNPs, Cit-modified Ir(III)@SiNPs and Ir (III)@SiNPs-Eu3+ under pH = 7.0 are presented in Fig. 1D. NH2groups are covalently grafted onto the surface of Ir(III)@SiNPs using APTES, leading to NH2-modified Ir(III)@SiNPs with higher potential than Ir(III)@SiNPs. Cit-modified Ir(III)@SiNPs possess obviously lower potential than NH2-modified Ir(III)@SiNPs, which is expected because carboxyl groups are covalently grafted onto Ir(III)@SiNPs surface using Cit. Via the chelation of Eu3+ with Cit, the potential of Ir(III)@SiNPsEu3+ is neutral, which further confirms the synthesis in Scheme S1. The FTIR spectra of SiNPs also record the changes after structural modifications. NH2-modified SiNPs exhibits a broad NeH bending vibration with the maximal peak at 1554 cm–1 (Fig. S3, a), confirming the presence of amino groups (Li et al., 2017). The peaks at 1145 and 1058 cm–1 are attributed to the strong vibration of SieOH. Ir(III) complex 1 shows the characteristic peak of the CeH vibrations in benzene and pyridine rings at 817 cm−1 which belongs to the ligands of complex 1 (Fig. S3, b). The covalent grafting between eNH2 on the surface of SiNPs with the eCOOH in Cit is confirmed by the peak at 1557 cm–1, which is attributed to COeNH vibrations. The success doping of Ir(III) complex 1 can be verified by the presence of the band at 800 cm–1 (Fig. S3, c). After coordination with Eu3+, the weakened peak at 3447 cm–1 (Fig. S3, d) indicated that Eu3+ has successfully coordinated onto the nanoparticle. The luminescence decay rates of Ir (III) complex 1, Ir(III)@SiNPs, Ir(III)@SiNPs-Eu3+ and Ir(III)@SiNPsEu3+/TC are measured by time-correlated single photon counting technique at 505 nm (Fig. S4). The luminescence lifetime of complex 1 is determined as 1.91 μs from the decay curve, while the lifetime of Ir (III)@SiNPs is 4.12 μs, which is longer than that of complex 1 because the silica environment partially protects the excited state of complex 1 from oxygen or water quenching (Kesarkar et al., 2017). The luminescence lifetime of Ir(III)@SiNPs-Eu3+ is 4.22 μs, which is close to the lifetime of Ir(III)@SiNPs and indicates that there is no obvious effect on the emission of Ir(III)@SiNPs upon the coordination with Eu3+ ions. In the presence of TC, the luminescence lifetime of Ir(III)@SiNPs-Eu3+ is

2.4. Determination of TC in buffer solution TC was added to the Tris-HCl (10 mM, pH = 9.0) buffer solution containing 1 mg mL–1 of Ir(III)@SiNPs-Eu3+. After 1 min, the fluorescence spectra were recorded under the excitation at 310 nm. A variety of TC concentrations were investigated and and the final concentrations of TC were 0, 0.01, 0.06, 0.1, 0.4, 0.6, 0.8, 1, 4, 6, 8, 10, 14, 16, 18 and 20 μM, respectively. 2.5. Determination of Hg2+ in buffer solution Ir(III)@SiNPs-Eu3+/TC complex (1 mg mL–1 of Ir(III)@SiNPs-Eu3+ and 20 μM of TC) was further used to detect Hg2+. In the detection, the final concentrations of Hg2+ were 0, 0.3, 0.7, 1, 3, 5, 7, 9, 11, 13 and 15 μM, respectively. The selectivity experiment was performed through adding 10 μL of other metal ions (Na+, K+, Zn2+, Ca2+, Ce3+, La3+, Zr4+, Mn2+, Ba2+, Co2+, Fe2+, Mg2+, Pb2+, Fe3+ and Cu2+, respectively) to the detection solution, while the interference experiment was carried out through adding the mixture of Hg2+ with other metal ions. The final concentration of interfering metal ions was 15 μM, and the total volume of the system was 500 μL. The sample solutions were respectively incubated for 1 min at room temperature, and the emission spectra were recorded under excitation at 310 nm. 2.6. Determination of TC in human serum through TRES 25 μL human serum was added into the detection system, and the other detection conditions and procedures were the same as the detection carried out in buffered solution. TRES were measured by a Horiba Fluorolog TCSPC spectrophotometer. TRES measurement spectra were recorded with the wavelength range of 350–680 nm and the time range of 0–20 μs (with the interval of 10 ns) under the excitation wavelength of 310 nm. In this testing, the TRES presents a discontinuous spectrum with the interval of 10 nm. Hence, 510/620 nm are selected as the emission wavelengths of Ir(III)@SiNPs-Eu3+ since they are the nearest wavelengths to the maximal emission peaks (505/ 616 nm) in steady state emission spectrum (SSES). 3. Results and discussion 3.1. Characterization of Ir(III)@SiNPs-Eu3+ The synthesis procedure of Ir(III)@SiNPs-Eu3+ and the structure of Ir(III) complex 1 were presented in Scheme 1. Ir(III)-doped SiNPs were first functionalized with APTES to fabricate NH2-modified Ir(III)@ SiNPs, which was then reacted with Cit to synthesize coordination pockets for Eu3+. Then, the morphology and size distribution of the assynthesized Ir(III)@SiNPs-Eu3+ were examined by TEM and dynamic

Scheme 1. The synthetic route of Ir(III)@SiNPs-Eu3+. 3

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Fig. 1. (A) and (B) TEM overview images of Ir (III)@SiNPs-Eu3+. The inset shows the particle size distribution histogram. (C) Survey XPS spectra of NH2-modified SiNPs and Ir(III)@ SiNPs-Eu3+. (D) Zeta potential of (a) Ir(III)@ SiNPs without modification, (b) NH2-modified a, (c) Cit-modified b and (d) Ir(III)@SiNPsEu3+ (that is, Eu3+-modified c) All of the particles were dispersed in water.

also 4.22 μs, showing that the luminescent quenching of Ir(III)@SiNPsEu3+ at 505 nm by TC is IFE, not fluorescence resonance energy transfer (FRET).

luminescence at 611 nm of Ir(III)@SiNPs-Eu3+. Meanwhile, the UV absorption of TC is wide and covers the excitation spectrum of Ir(III) complex, so the green luminescence at 505 nm of Ir(III)@SiNPs-Eu3+ is quenched though the IFE of TC to complex 1. According to the reported stoichiometry of 2:1:1 for Cit, Eu3+ and TC (Lin et al., 2004), a molecule structure involved recognition of TC is also proposed in Scheme S1. Therefore, the TC recognition mechanism of Ir(III)@SiNPs-Eu3+ is based on the IFE between TC with Ir(III) complex and the AETE from TC to Eu3+ in Ir(III)@SiNPs-Eu3+.

3.2. Principle of TC detection by using Ir(III)@SiNPs-Eu3+ Fig. 2A exhibits that the emission of Eu3+/TC is very weak due to the vibration of the coordinated water molecules. Upon the addition of Cit-modified Ir(III)@SiNPs, the emission intensity of Eu3+/TC increases significantly, showing that Cit coordinates with Eu3+. The emission band at 505 nm is attributed to the Ir(III) complex 1 (Hu et al., 2018), while the emission bands at 579, 591, 616, 652 nm are attributed to Eu3+ characteristic transition from 5D0 to 7F0−3 (Xu et al., 2012). Fig. 2B shows that the wide UV absorption (around 270 nm) of TC covers the excitation of Ir(III)@SiNPs-Eu3+, so IFE can happen to quench the luminescence of Ir(III)@SiNPs-Eu3+. The recognition mechanism of Ir(III)@SiNPs-Eu3+ nanoprobe to TC is schematically presented in Scheme 2. Upon the addition of TC, a ternary complex forms between TC, Eu3+ and Cit groups which has already been anchored on the surface of Ir(III)@SiNPs. TC contains a β-diketonate configuration, which easily coordinates with Eu3+ and forms into stable TC/Eu3+ complex and thus the energy of TC is transfered to Eu3+ through adsorption energy transfer emission (AETE). This greatly enhances the red

3.3. Optimization of Ir(III)@SiNPs-Eu3+ for TC detection Fig. S5A–C shows the effects of the concentrations of Eu3+and Ir(III) @SiNPs-Eu3+, and pH on the relative emission intensity of F616/F505. As shown in Fig. S5A, with the addition of 20 μM TC, the relative emission intensity F616/F505 of the system first increases and then decreases with the increased Eu3+ concentration from 0–20 mM. The maximum relative intensity of the system appears at 10 mM of Eu3+. The relative emission intensity of F616/F505 varies with the concentration of Ir(III)@SiNPs-Eu3+ and reaches a maximal value at the concentration of 1 mg mL−1 (Fig. S5B). pH also has an effect on the luminescence of Ir(III)@SiNPs-Eu3+/TC complex, and the highest F616/ F505 value appears at pH = 9 (Fig. S5C). The high pH can cause the Fig. 2. (A) The excitation and emission spectra of Eu3+/TC complex under different conditions. (a) the excitation of Eu3+ + TC, (b) the emission of Eu3+ + TC, (c) the excitation spectra of Eu3+ + TC + Ir(III)@SiNPs (1 mg mL−1), (d) the emission of Eu3+ + TC + Ir(III)@ SiNPs (1 mg mL−1). (B) The spectra of TC and Ir(III)@ SiNPs-Eu3+. (a) UV–vis absorption spectrum of TC, (b) excitation of Ir(III@SiNPs-Eu3+, (c) emission spectra of Ir (III)@SiNPs-Eu3+.

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Scheme 2. Schematic illustration of TC detection based on Ir(III)@SiNPs-Eu3+ nanoprobe.

relationship, the limit of detection (LOD) for TC detection is calculated as 4.9 × 10–3 μM (3σ, n = 6) and its limit of quantification (LOQ) was also calculated as 1.6 × 10–2 μM (10σ, n = 6). The LOD is much lower than the allowable concentration (0.225 μM) specified by the European Union and China (Luo et al., 2015). Table S1 demonstrates that the LOD and the linear range of this nanoprobe are comparable or even superior to those reported fluorescent TC detection strategies. Additionally, the presence of different concentrations of TC can be discriminated through naked-eyes (Fig. 3C), demonstrating that this nanoprobe can be fabricated into visual TC detection in the forms of portable smart phone or test strips.

formation of europium hydroxide precipitate (Tan and Chen, 2012) and thus the decrease in the relative emission intensity of F616/F505. Therefore, 10 mM Eu3+, 1 mg mL−1 Ir(III)@SiNPs-Eu3+ and pH = 9.0 were selected as the optimal detection conditions in this study. 3.4. Performance of the nanoprobe for TC detection Under the optimal detection conditions, the performance of Ir(III)@ SiNPs-Eu3+ for TC assay is investigated through titration experiment. As shown in Fig. 3A, Ir(III)@SiNPs-Eu3+ exhibits a moderate emission band at 505 nm in the absence of TC when excitated at 310 nm, and the emission of Eu3+ is not observed. Once TC is added to the solution in the concentration range of 0 − 20 μM, the luminescence intensity of the nanoprobe at 616 nm gradually increases, while the emission intensity at 505 nm gradually drops down. The relative emission intensity of F616/F505 has excellent linear relationship with the TC concentration in the range of 0.01 − 20 μM (Fig. 3B). The linear relationship is described as F616/F505 = 0.3438CTC − 0.07825 with a correlation coefficient of 0.9915, where “CTC” is the concentration of TC. Based on this linear

3.5. Selectivity and anti-interference ability of Ir(III)@SiNPs-Eu3+ for TC detection In order to investigate the influence of some coexisted substances in biological systems, including amino acids, glutathione (GSH), glucose and metal ions, on the detection performance of the nanoprobe, these substances are selected as potential interfering samples to evaluate the Fig. 3. (A) Luminescent responses of the nanoprobe towards TC with various of concentrations (0, 0.01, 0.06, 0.1, 0.4, 0.6, 0.8, 1, 4, 6, 8, 10, 14, 16, 18 and 20 μM) in Tris/ HCl buffer. (B) The relationship of relative intensity F616/ F505 and the concentrations of TC (0, 0.01, 0.06, 0.1, 0.4, 0.6, 0.8, 1, 4, 6, 8, 10, 14, 16, 18 and 20 μM) in Tris/HCl buffer. Error bars represent the standard deviations (SD) of the results from three independent experiments. (C) Photographs of the detection system in the presence of TC with different concentrations (a: 0, b: 0.5, c: 1, d: 3, e: 5, f: 10, g: 15 and h: 20 μM) under the illumination of UV lamp at 305 nm.

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Fig. 4. (A) The SSES of the 5 % (v/v) human serum contained detection system in the absence (black line) or presence (blue line) of 20 μM TC. (B) The TRES of the 5 % (v/v) human serum contained detection system in the absence (black line) or presence (blue line) of 20 μM of TC. (C) The TRES responses of the detection system containing 5 % (v/v) human serum towards to TC with various concentrations (1, 5, 10, 15 and 20 μM). (D) Linear plot of the relative luminescence intensity at F620/F510 towards TC concentrations in the detection system containing 5 % (v/v) human serum.

specificity and anti-interference of Ir(III)@SiNPs-Eu3+ for TC detection. As shown in Fig. S6, aspartic acid (Asp), proline (Pro), glutamic acid (Glu), histidine (His), lysine (Lys), GSH, Zn2+, Ca2+, K+, Mg2+, Ba2+ and glucose do not bring influence on the F616/F505 value of Ir(III)@ SiNPs-Eu3+, and only TC or their mixtures with TC induces significant changes in the value of F616/F505 at the same concentration. These results indicate that Ir(III)@SiNPs-Eu3+ possesses high selectivity and strong anti-interference ability for TC detection in complicated systems.

Ce3+, La3+, Zr4+, Mn2+, Ba2+, Co2+, Fe2+, Mg2+, Pb2+ and Fe3+ do not lead to obvious luminescence quenching. The co-existence of these ions with Hg2+ in the solution also do not bring any influence to Hg2+induced luminescence quenching, which indicates good selectivity and anti-interference of Ir(III)@SiNPs-Eu3+/TC towards Hg2+ over other metal ions. Although Cu2+ shows obvious luminescence quenching of the system, the resulting luminescence intensity was almost two times as that of Hg2+, indicating the weaker quenching performance of Cu2+ than Hg2+. In addition, the Cu2+ sequestering agent, such as KCN, can be used to remove the interference of Cu2+ (Fig. S8). According to the optimization results, the conditions of pH = 9.0, 20 μM of TC and 1.0 mg mL−1 of Ir(III)@SiNPs-Eu3+ provide the optimal detection of Hg2+ (Fig. S9A–C). The luminescence quenching kinetics of Ir(III)@ SiNPs-Eu3+/TC complex is further studied through the luminescence titration with various concentrations of Hg2+. In Fig. S10A, Ir(III)@ SiNPs-Eu3+/TC complex shows a stronger emission at 616 nm and a moderate emission at 505 nm, while its emission at 616 nm gradually drops down and the emission at 505 nm remains unchanged after the addition of 0.3–15 μM of Hg2+. A prominent linear relationship between the luminescence intensity and the Hg2+ concentrations is observed in the concentration range of 0.3–9 μM (Fig. S10B). Accordingly, the detection limit for Hg2+ assay was calculated as 3.66 × 10–2 μM. Therefore, the Ir(III)@SiNPs-Eu3+/TC complex can be used for the sensitive relay recognition of Hg2+ in a ratiometrically luminescent mode.

3.6. Principle and performance of Hg2+ detection by using Ir(III)@SiNPsEu3+/TC Hg2+ as one of the most toxic heavy metal pollutant, not only causes environmental pollution, but also has a negative effect on human body (Poornima et al., 2016; Wu et al., 2017; Wang et al., 2012). Due to the strong interaction between Hg2+ and Cit (the binding constant is as high as K=1010.90) (Singh et al., 1996), the recognition of Hg2+ by complex Ir(III)@SiNPs-Eu3+/TC is further investigated. As shown in Fig. S7, Ir(III)@SiNPs-Eu3+ shows only one signal decrease at 505 nm, while Ir(III)@SiNPs-Eu3+/TC exhibits constant signal at 505 nm and decreased signal at 616 nm, indicating the ratiometric detection of Hg2+. In addition, at the same concentration (3 μM) of Hg2+, the luminescence of Ir(III)@SiNPs-Eu3+/TC at 616 nm was decreased by 45 %, while the luminescence of Ir(III)@SiNPs-Eu3+ at 505 nm was just decreased by 34 %, showing lower response performance. Hence, Ir(III) @SiNPs-Eu3+/TC nanoprobe possesses superior detection properties for Hg2+. Based on this experimental result, the principle of Hg2+ detection using Ir(III)@SiNPs-Eu3+/TC as probe is illustrated in Scheme S2. The detection of Hg2+ is based on the displacement of Eu3+ from the complex of Cit-Eu3+-TC by the newly added Hg2+ because of the stronger binding affinity (the binding constant is as high as K=1010.90) of Cit to Hg2+ than that of Cit-Eu3+-TC, the dissociation constants (pKd) of which was reported in the range of 4.2–4.9 (Lin et al., 2004). Upon the addition of Hg2+, it displaces Eu3+ from Ir(III)@SiNPs-Eu3+/TC to coordinate with Cit, and Eu3+ is released into the solution to be solvated by water molecules, resulting in the great quenching of its luminescence. Meanwhile, the luminescence intensity of the Ir(III) complex in the SiNPs is not changed during this process. Therefore, the ratiometric luminescent changes of Ir(III)@SiNPs-Eu3+/TC can be utilized for the detection of Hg2+. Fig. S8 shows other mental ions, such as, Na+, K+, Zn2+, Ca2+,

3.7. Detection of TC in human serum through TRES An enormous signal occurs in the steady state emission spectrum (SSES) in a detection system containing 5 % (v/v) human serum at 370 nm, which is caused by the endogenous fluorophores contained by human serum (Fig. 4A). Although the emission of Ir(III) complex 1 locates at 505 nm, the luminescent responses of the system to 20 μM of TC is heavily muted by the strong background signal of human serum, leading to failure of the detection. In virtue of the long lifetime property of Ir(III)@SiNPs-Eu3+, TRES is used to reduce the high fluorescent interference of the serum. Through recording the luminescent signals of the detection system after the excitation for 4.5 μs, the TRES responses of the detection system to 20 μM TC is clearly observed and makes the detection of TC feasible in serum. (Fig. 4B). The TRES intensities of the system grow with the increased concentrations of TC (1, 5, 10, 15 and 6

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Fig. 5. Luminescence micrographs of LLCPK15 cells under different conditoins. (A) Luminescence image of LLC-PK15 incubated with Ir(III)@SiNPs-Eu3+ (100 μg mL−1) in green channel, (B) Luminescence image of A in red channel, (C) Bright field image of A and B, (D) Luminescent image of LLC-PK15 incubated with Ir(III)@SiNPs-Eu3+ (100 μg/mL) for 6 h, and then further incubated with TC (50 μM) from green channel, (E) Luminescentce image of D from red channel, (F) Bright field image of D and E, (G) Luminescent image of LLC-PK15 incubated with Ir(III)@SiNPs-Eu3+ (100 μg mL−1) for 6 h, and then further incubated with TC (50 μM) and Hg2+ (50 μM) in sequence in green channel. (H) Luminescent image of G in red channel, (I) Bright field image of G and H.

20 μM) (Fig. 4C). A good linear relationship between the relative emission intensity and the concentration of TC is obtained (R2 = 0.9910) from 1 to 20 μM of TC (Fig. 4D).

experiment has been performed in lake water samples. As shown in Table S2, the recoveries of water samples are obtained from 98.9% to 112.0%, and the RSD is below 2.25 %. These results suggest that the developed nanoprobe possesses high potential application in food safety and environmental monitoring.

3.8. Cytotoxicity and cell imaging of Ir(III)@SiNPs-Eu3+ The cytotoxicity of Ir(III)@SiNPs-Eu3+ is studied before cell imaging. As shown in Fig. S11, the porcine renal cells have no obvious changes in the presence of Ir(III)@SiNPs-Eu3+ and their viability remains higher than 90 % even when the concentration of Ir(III)@SiNPsEu3+ is up to 200 μg mL−1, confirming the low cytotoxicity of Ir(III)@ SiNPs-Eu3+. In the cell imaging experiment, the cells exhibit strong green luminescence in green channel (Fig. 5A) but almost no luminescence in red channel (Fig. 5B), indicating that the cells are infected successfully by Ir(III)@SiNPs-Eu3+. Then, the cells are subsequently incubated with TC, the high luminescence in green channel almost disappears (Fig. 5D) and enhanced luminescence in the red channel appears (Fig. 5E), indicating the coordination of TC with Ir(III)@SiNPsEu3+ in the cells. Lastly, after Hg2+ is added into the incubation solution, the luminescence in green channel almost keep unchanged (Fig. 5G), while the luminescence of the cell in red channel is quickly quenched (Fig. 5H). This phenomenon further verifies the preferential coordination between Hg2+ with Cit, resulting in the decomposition of Ir(III)@SiNPs-Eu3+/TC complex and the release of Eu3+. In addition, Fig. 5C, F and 5I present the white-light imaging of LLC-PK15 cells, which are treated by Ir(III)@SiNPs-Eu3+, Ir(III)@SiNPs-Eu3+/TC and Ir (III)@SiNPs-Eu3+/TC + Hg2+, respectively, demonstrating that these cells keep intact during the whole of cell imaging. These results exhibit that Ir(III)@SiNPs-Eu3+ also possesses prominent recognition ability to TC and Hg2+ in living cells.

4. Conclusion In summary, a novel ratiometrically luminescent TC nanoprobe Ir (III)@SiNPs-Eu3+ has been designed and fabricated. Ir(III)@SiNPs is prepared through doping Ir(III) complex into SiNPs by the modified stober method. APTES and Cit are in turn covalently grafted onto the surfaces of SiNPs to endow pockets for Eu3+ coordination. The complexation of TC with Eu3+ in Ir(III)@SiNPs-Eu3+ makes Eu3+ emit a strong luminescence signal at 616 nm through AETE, while the strong luminescence of Ir(III)@SiNPs is selectively quenched due to the IFE of TC. The developed nanoprobe exhibits a sensitive response with a linear range of 0–20 μM and a detection limit of 4.9 × 10–3 μM of TC. More importantly, the nanoprobe demonstrates its advantages for TC detection in complicated system and the distinct cell imaging for TC monitoring, providing a new long-lived luminescent nanoprobe for TC detection in real samples. The Ir(III)@SiNPs-Eu3+ can also be further used for the relay recognition of Hg2+ over other competing metal ions with excellent sensitivity, selectivity and anti-interference ability. The cytotoxicity of Ir(III)@SiNPs-Eu3+ is very low and thus the detection for TC and Hg2+ also works well in living cells via cell imaging. The developed nanoprobe has the advantages of long lifetime, striking luminescent color changes, excellent anti-interference ability, displaying high application potential in biological systems. In addition, the luminescence and the sensitivity of the nanoprobe can be further improved by doping other metal complexes with excellent photophysical properties or designing novel nanomaterials for nanoprobe fabrication.

3.9. Real sample detection of Ir(III)@SiNPs-Eu3+

CRediT authorship contribution statement

In order to further confirm the reliability of the ratiometric nanoprobe, TC detections were carried out in milk and lake water samples, respectively. The titration experiment in milk samples showed that the luminescence intensity of Ir(III)@SiNPs-Eu3+ positively increases as the concentrations of TC grow in the range of 0.01–14 μM (Fig. S12A and B) and the LOD is estimated as 8.7 × 10–3 μM. The recovery

Xiaotong Li: Conceptualization, Methodology, Writing - review & editing. Kaimei Fan: Methodology, Software, Data curation. Ruimei Yang: Resources, Investigation. Xiuxiu Du: Investigation. Baohan Qu: Supervision. Xiangmin Miao: Writing - review & editing. Lihua Lu: 7

Journal of Hazardous Materials 386 (2020) 121929

X. Li, et al.

Funding acquisition, Supervision, Writing - review & editing.

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