Development of a Luminescent Dinuclear Ir(III) Complex for

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Development of a Luminescent Dinuclear Ir(III) Complex for Ultrasensitive Determination of Pesticides Lihua Lu, Huijuan Su, Qingyun Liu, and Feng Li Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b03687 • Publication Date (Web): 07 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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Analytical Chemistry

Development of a Luminescent Dinuclear Ir(III) Complex for Ultrasensitive Determination of Pesticides Lihua Lu,†,§ Huijuan Su,†,§ Qingyun Liu,‡ and Feng Li*,† †College of Chemistry and Pharmaceutical Sciences, Qingdao Agricultural University, Qingdao 266109, People’s Republic of China ‡College of Chemical and Environmental Engineering, Shandong University of Science and Technology, Qingdao 266510, China *Corresponding author. Tel/Fax: 86-532-86080855, E-mail: [email protected]. §These authors contributed equally to this work.

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ABSTRACT

To improve the G-quadruplex specificity of Ir(III) complexes, a novel dinuclear Ir(III) complex (Din Ir(III)–1) was designed and synthesized through connecting two mononuclear Ir(III) complexes via a diphenyl bridge. Din Ir(III)–1 presents 3.4–4.1 fold enhancements for Gquadruplex relative to ssDNA and 4.3–5.3 fold enhancements relative to dsDNA in luminescence intensity, respectively, demonstrating an excellent G-quadruplex selectivity. Ascribed to its superior specificity to G-quadruplex, Din Ir(III)–1 was employed to construct a highly sensitive luminescent pesticides detection platform. The detection is based on acetylcholinesterase (AChE)-catalyzed hydrolysis product-induced DNA conformational transformation and subsequent terminal deoxynucleotidyl transferase (TdT) directed G-quadruplex formation. The assay exhibited a linear response between the emission intensity of Din Ir(III)–1 and the pesticide concentration in the range of 0.5 to 25 µg/L (R2 = 0.994), and the limit of detection for the pesticide was as low as 0.37 µg/L when using aldicarb as the model pesticide. Moreover, this strategy demonstrates good applicability for the pesticide detection in real samples. It is also versatile for the detection of other organophosphate or carbamate pesticides which have the inhibition ability towards AChE. Therefore, the proposed approach is scalable for practical application in food safety and environmental monitoring fields and will provide promising solutions for the assay of pesticide residues.

KEYWORDS: dinuclear Ir(III) complex; pesticide detection; G-quadruplex; terminal deoxynucleotidyl transferase; luminescence


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INTRODUCTION Organophosphorous (OPs) and Organocarbamate (OCs), such as aldicarb, carbaryl, diazion, parathion, have been extensively used as pesticides in crops and plants to achieve higher yields due to their lethality towards insects and pests. However, abusing these pesticides often causes overmuch residue which results in prodigious effects to human health and environment because of their extremely high toxicity.1-3 The toxicity of OPs and OCs mainly comes from their strong ability to inhibit the activity of acetylcholinesterase (AChE) enzyme. AChE is able to hydrolyze acetylcholine into choline and acetate, so the presence of pesticides can directly cause overdose of acetylcholine in living organisms and heavily affects the physiology of the nervous system.4,5 It is therefore highly important to explore judicious methods to sensitively determine minimal level pesticide residues in environmental and agricultural products to prevent the assimilation of pesticides by humans. Although gas chromatography (GC),6 high-performance liquid chromatography (HPLC),7 enzyme-linked immunosorbent assay (ELISA),8,9 and surface plasmon resonance (SPR)10,11 have been widely employed to monitor pesticide contamination, these techniques tend to need expensive instrumentation, tedious sample preparation procedures and specially trained operators. Meanwhile, AChE enzyme-based biosensors have been developed in recent years as the most promising alternative strategy in pesticides detection ascribed to their fast response, high sensitivity, low cost, and easy on-site analysis. The AChE enzyme inhibition property of OPs and OCs has been turned into electrochemical,12-17 electrochemiluminescent,18,19 colorimetric20-25 and luminescent20,26-36 signals in various pesticides detection. Among them, luminescent strategy is more attractive for pesticide assay ascribed to its promising advantages of high sensitivity and simplicity.


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Cyclometallated iridium(III) (Ir(III)) complexes have attracted great interest as luminescent probes in virtue of their long lifetime, large Stokes shift, low cytotoxicity, simple synthetic procedures as well as tunable excitation and emission maxima over the visible light region.37-39 Recent years, Ma’s group has found several mononuclear Ir(III) complexes which can act as Gquadruplex-selective probes for the development of label-free luminescent biosensors.40 As reported that mononuclear Ir(III) complex potentially interact with the loop parts of Gquadruplex through non-covalent interactions,41,42 we conjectured that dinuclear Ir(III) complexes prepared by connecting two mononuclear Ir(III) complexes may increase the binding sites between dinuclear Ir(III) complexes with G-quadruplex, which can enhance the noncovalent interactions of the binding event, and thus strengthen the binding ability of the designed dinuclear Ir(III) complexes. This enhanced binding would be expected to increase the luminescent signal amplification and recognition and thus greatly increase the detection sensitivity. A dinuclear Ir(III) complex [Ir2(tpptda)(phq)4]+ was recently obtained through connecting two molecules of mononuclear Ir(III) complex via a simple five-carbon bridge linkage, which has been successfully used in the detection of transcription factor.43 Considering the infancy development of dinuclear Ir(III) complexes and its inspiring performance in Gquadruplex-based detection, it is highly desirable to explore new dinuclear Ir(III) complexes, which could not only be able to pursue G-quadruplex-specific probes with higher sensitivity, but also to provide promising alternatives for the construction of label-free luminescent sensing platforms for OPs or OCs assays in food safety or environmental monitoring fields. Terminal deoxynucleotidyl transferase (TdT) is a DNA polymerase that catalyzes the polymerization at the 3′-OH ends of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) with protruding or blunt terminals in a template-free mode.44-48 Accordingly, the


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resulted sequence is usually random and mainly depends on the contents of polymerization deoxyribonucleoside triphosphate (dNTP) pool. As reported, G-quadruplex-forming sequences can be randomly generated by TdT when the dNTP pool contains 40% dATP and 60% dGTP, and the percentage of guanine (G), which participates in a 20-nucleotide G-quadruplex structure containing four G-tracts, is thought to be about 50−70%.49-51 Inspired by these concepts, we intended to develop a novel luminescent dinuclear Ir(III) complex for ultrasensitive determination of pesticides based on AChE-mediated DNA conformational switch. In virtue of its unique property in G-quadruplex generation, TdT will be acted as signal transducer and amplifier in this detection. To our knowledge, no luminescent dinuclear Ir(III) complex-based assay for the detection of pesticides has yet been reported. 52-55 EXPERIMENTAL SECTION Materials. Iridium chloride hydrate (IrCl3.xH2O) was purchased from Sigma Aldrich (St. Louis, MO). Acetylcholinesterase (AChE) and acetylthiocholine iodide (ATCh) were obtained from Aladdin Industrial Corporation (Shanghai, China). TdT was obtained from New England Biolabs Ltd. (Beijing, China). Oligonucleotide, dATP, dGTP and dNTP were offered by Sangon (Shanghai, China). The DNA sequences used in this work were listed in Table S1. Synthesis of Din Ir(III)–1. The N^N ligand 4,4'-di(1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)biphenyl (named as L–1) was prepared according to the reported method.56 The Ir(III) complex dimer [Ir2(ppy)4Cl2] was also synthesized according to the literature’s method.57 Then, a suspension of [Ir2(ppy)4Cl2] (0.2 mmol) and newly prepared L–1 (0.2 mmol) were added in a mixture of DCM/methanol (1:1, 20 mL), and refluxed under the protection of nitrogen. After 20 h’s reaction, a methanol solution of ammonium hexafluorophosphate (excess) was dropwised


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into the gradual transparent reaction solution. Then, the mixture was distilled by rotary evaoration until the orange precipitation appeared. To filter and wash the precipate with several portions of water and diethyl ether, crude product was received, which was further purified by recrystallization in acetonitrile/diethyl ether vapor diffusion to get the titled complex Din Ir(III)– 1. Complex Din Ir(III)–1. Yield: 66%. 1H NMR (500 MHz, Acetone-d6) δ 9.35-9.24 (m, 4H), 8.538.44 (m, 4H), 8.36-8.33 (m, 4H), 8.25 (s, 4H), 8.04 (s, 2H), 7.96 (d, J = 6.8 Hz, 6H), 7.90 (d, J = 8.0 Hz, 6H), 7.76-7.73 (m, 8H), 7.10 (d, J = 6.4 Hz, 4H), 7.00 (s, 8H), 6.47 (s, 4H);

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C NMR

(100 MHz, Acetonitrile-d3) δ 150.9, 150.1, 149.7, 145.4, 144.9, 139.1, 132.7, 132.3, 131.0, 130.7, 128.1, 127.9, 127.4, 125.5, 124.0, 123.2, 120.4; MALDI-TOF-HRMS: Calcd. for C82H54Ir2N12[M–PF6]+ : 1592.3853 Found: 1592.3791. Mon Ir(III)–2. Reported. 58 Detection of pesticides in aqueous solution. Firstly, 1 µM of DNA probe (DP) and 3 µM of Hg2+ ions were mixed to form three T−Hg2+−T base pairs in each DNA probe molecule. Various concentrations of aldicarb and 1 mU/mL of AChE were incubated in Tris–HCl buffer solution (10 mM Tris, 10 mM MgCl2 and 100 mM NaCl, pH = 8.0) for 30 min at 37 °C. Then, 30 µM of acetylthiocholine chloride (ATCh) was added into the mixture and further incubated at 37 °C for 100 min. Next, the typical enzymatic generation of G-rich random sequences by TdT was carried out in TdT reaction buffer (0.2 M potassium cacodylate, 0.025 M Tris, 0.01% (v/v) Triton X-100, pH = 7.2) at 37 °C. The polymerization reaction solution contained 0.2 mM dATP, 0.3 mM dGTP, 30 U TdT, 0.25 mM CoCl2 and certain volume of TdT reaction buffer. Two hours later, the polymerization reaction was terminated by heating the solution at 75 °C for 10 min. Then, 50


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mM KCl was inputted into the reaction solution to promote the formation of G-quadruplex. Finally, 2 µM of Din Ir(III)–1 was added into the prepared samples before the emission detection and the total volume was 100 µL. All of the concentrations mentioned in these procedures are based on the final volume of 100 µL, and the final detection system contains 0.5 µL acetonitrile which is not included in the final volume of 100 µL. RESULTS AND DISCUSSION Dinuclear Ir(III) complex 1 (Din Ir(III)–1) (Figure 1A) carries the C^N ligand 2phenylpyridine (ppy) and N^N ligand which was prepared through condensation reaction between two equivalents of 1,10-phenanthroline-5,6-dione with one equivalent of biphenyl-4,4'dicarbaldehyde in the mixture of ammonium acetate/glacial acetic acid (Figure S1). From the perspective of structure, Din Ir(III)–1 is the dimer of mononuclear Ir(III) complex 2 (Mon Ir(III)–2) (Figure 1B). The photophysical properties of Din Ir(III)–1 were presented in Table S2. In the initial research, although Mon Ir(III)–2 was found to demonstrate certain specificity towards G-quadruplex, its rather poor selectivity to G-quadruplex causes that it could not be used as a G-uadruplex-selective probe to establish a sensitive biosensor. It is reported that mononuclear Ir(III) complexes potentially interacted with the loop regions of the G-quadruplex, so we envisaged to synthesize the dimer of Mon Ir(III)–2 to enhance the interaction between loops of G-quadruplex and Ir(III) complex, and thus to improve the metal complexes’ specificity to G-quadruplex. To our delight, the prepared Din Ir(III)–1 shows obvious enhanced selectivity to G-quadruplex. Four kinds of G-quaruplex-forming DNA including c-myc, c-kit87, PS2.M and Pu27, were used to verify the selectivity of Ir(III) complexes. Figure 2A showed that Din Ir(III)– 1 exhibited enhanced luminescence signal towards all of the tested G-quadruplexes, but just slight signal changes were observed for ssDNA and dsDNA. A typical G-quadruplex-based


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detection platform usually involves the conformational changes of the designed DNA probe, for example, from ssDNA to G-quadruplex, or from dsDNA to G-quadruplex. Hence, as a Gquadruplex probe, its ability of recognizing a G-quadruplex from the existed ssDNA or dsDNA is pivotal and determines the discrimination ability and sensitivity of the designed G-quadruplex probe. “IG4/Iss” is used to indicate the selectivity of Ir(III) complex between G-quadruplex and ssDNA, and “IG4/Ids” presents the selectivity of Ir(III) complex to dsDNA. Figure 2C showed that Din Ir(III)–1 possesses 3.4–4.1 fold change for G-quadruplex relative to ssDNA and 4.3–5.3 fold change relative to dsDNA, indicating an excellent G-quadruplex selectivity. In contrast, Mon Ir(III)–2 just showed a relatively lower specificity to G-quadruplex (Figure 2B), and presented the values of “IG4/Iss” in the range of 0.6–0.9 and “IG4/Ids” in the range of 1.1–2.4 (Figure 2D), demonstrating a poor discrimination ability. The specific binding of Din Ir(III)–1 to G-quadruplex structure was further verified by Gquadruplex fluorescent intercalator displacement (G4-FID) experiments. The G4-FID assay was used to verify the G-quadruplex-binding ability of a G-quadruplex probe through analyzing the thiazole orange (TO) displace ability of the tested molecules from TO/G-quadruplex complex. Figure S2 showed that Din Ir(III)–1 can replace thiazole orange (TO) from TO/G-quadruplex complex with a

G4

DC50 value (half-maximal concentration of compound required to displace

50% TO from TO/DNA hybrid) of 5.5 µM. By contrast, even used the highest concentrations of Din Ir(III)–1 in the experiment, less than 50% of TO was displaced from dsDNA/TO hybrid. These results surely indicate that Din Ir(III)–1 prefers to bind with G-quadruplex structure over double-stranded motif. In order to investigate the interaction between Din Ir(III)–1 with Gquadruplexes, the loop-effect experiment was carried out. The luminescent responses of Din Ir(III)–1 towards a series of G-quadruplexes (5′-TG3TAG3TnG3T2G3-3′) containing different


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loop parts with the size ranging from 1 to 15 nucleotides (nt). The results demonstrated that the luminescent intensity of Din Ir(III)–1 rise with a longer loop size (Figure S3), suggesting that dinuclear Ir(III) complexes still bind with G-quadruplex at its loop region as that of mono nuclear Ir(III) complexes59,60 and the G-quadruplex loop still take an important role in the interaction between Din Ir(III)–1 with G-quadruplexes. Based on this result, we inferred that double binding sites in dinuclear Ir(III) complex could provide much more specific recognition capability for Din Ir(III)–1 to bind with G-quadrulexes’ loop part, affording it a higher selectivity towards G-quadrulex.

Figure 1. Chemical structures of Din Ir(III)–1 and Mon Ir(III)–2.


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Figure 2. A. The luminescent responses of Din Ir(III)–1 to different DNA structures (ssDNA: ss17; dsDNA: ds-17; G-quadruplex DNA: c-myc, c-kit87, PS2.M, Pu27) B. The luminescent responses of Mon Ir(III)–2 to different DNA structures. C. Diagrammatic bar array representation of the luminescence enhancement selectivity ratio of Din Ir(III)–1 for Gquadruplex DNA (c-myc, c-kit87, PS2.M, Pu27) over dsDNA (ds-17) or ssDNA (ss-17). D. Diagrammatic bar array representation of the luminescence enhancement selectivity ratio of Mon Ir(III)–2 for G-quadruplex DNA (c-myc, c-kit87, PS2.M, Pu27) over dsDNA (ds-17) or ssDNA (ss-17). Error bars represent the standard deviations of the results from three independent experiments. The proposed mechanism of the label-free detection of pesticides using luminescent Gquadruplex-selective Din Ir(III)–1 was presented in Scheme 1. The sensing system mainly involves with DP/Hg2+, ATCh, AChE, TdT and Din Ir(III)–1. The initial ssDNA DP (Table S2), containing six thymin (T) bases, can hybridize with three equivalents of Hg2+ to form a bluntended hairpin configuration by T–Hg2+–T mismatched base pairs. ATCh acts as the enzymatic substrate of AChE, since ATCh can be transduced into thiocholine (TCh) under the catalysis of AChE enzyme. Then, the thiol group (–SH) in TCh possesses the ability to sequester Hg2+ from the T–Hg2+–T base pairs ascribed to the higher affinity of –SH/Hg2+ than that of T–Hg2+–T mismatch. Once Hg2+ is captured by TCh from the hairpin-shaped DP, DP will be liberated into


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ssDNA, revealing its 3′-terminal, and thus TdT can generate series G-rich DNA sequences at this free 3′-terminal of DP in the presence of a 60% dGTP and 40% dATP pool. Then G-quadruplexselective probe Din Ir(III)–1 is able to recognize the newly generated G-quadruplex. Without aldicarb, AChE can catalyze ATCh into TCh, and the –SH group in TCh is able to take Hg2+ away from the as-prepared hairpin DP, leading to the generation of many unfolded DP with single-stranded configuration. Once single-stranded DP formed, TdT-directed G-rich DNA sequences are produced. The TdT polymerase reaction product then forms into a series of Gquadruplex motifs in the presence of K+. Upon the addition of Din Ir(III)–1, the G-quadruplex can bind with Din Ir(III)–1, leading to a significant luminescence enhancement. On the contrary, when pesticide aldicarb is added into the mixture, it quickly binds to AChE and inhibits its catalysis ability. Hence, less TCh is generated, resulting in less release of Hg2+-hybridized hairpin-structured DP. Consequently, almost all of the DP/Hg2+ maintained their initial hairpin configurations in the detection system. This hairpin structured DP/Hg2+ cannot be elongated by TdT due to their sequestered 3′-OH ends by T–Hg2+–T mismatch. Upon addition of Din Ir(III)–1, only a weak interaction between Din Ir(III)–1 with hairpin DP happened, resulting in a low luminescence signal. Employing the proposed “signal-off” strategy, luminescent label-free detection of pesticides can be readily realized.


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Scheme 1. Schematic diagram of the label-free pesticides detection based on a novel Gquadruplex-selective probe Din Ir(III)–1.

To verify the feasibility of the proposed strategy, the responses of Din Ir(III)–1 was investigated under different detection conditions. As shown in Figure 3A, no obvious luminescent signal was observed upon the addition of Din Ir(III)–1 into the buffered solution (curve a). When Hg2+-hybridized hairpin DNA DP and ATCh were added into the mixture, the system also showed a minimal emission signal (curve b), which was due to the weak interaction between Din Ir(III)–1 with the hairpin-structured DNA and the non-interference from ATCh. After incubating AChE with the mixture, there was still no obvious luminescence intensity appeared (curve c), which indicates no obvious interference from AChE. A strong enhancement in luminescence intensity was observed after the mixture is put into TdT, dATP and dGTP, showing that the G-quadruplex was generated in the presence of DP/Hg2+, ATCh, AChE, TdT and dNTP (curve d). As a contrast, when the mixture was incubated with aldicarb, the luminescent intensity showed a dramatic decrease. This demonstrates that aldicarb suppressed the G-quadruplex generation through inhibiting the activity of AChE, leading to obvious reduced luminescent signal of Din Ir(III)–1 (curve e). The gel electrophoresis experiment was carried out to further verify this detection (Figure 3B). The ssDNA DP was used as reference band (lane a) in this study. When DP hybridized with Hg2+, there is no obvious change in the band (lane b). The DP/Hg2+ hybrid was incubated with TdT, dATP and dGTP, showing a similar band as that by DP/Hg2+ alone, demonstrating that TdT cannot elongate the hybridized 3′-ends of DP/Hg2+ by T–Hg2+–T mismatch (lane c). After the addition of TdT, dATP and dGTP into the mixture of DP/Hg2+, ATCh and AChE, the system exhibited a much slower moving band (lane d) than the


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reference DP. According to the detection principle, this new band is the polymerization product of TdT. The generated band in this case showed that just in the presence of ATCh and AChE, TdT can hybridize dATP + dGTP onto the 3′-OH ends of DP ascribed to the production of TCh by ATCh and the grabbing Hg2+ by AChE from DP/Hg2+. When the same system was further treated by aldicarb, a similar band as that of DP was demonstrated (lane e), indicating that aldicarb inhibits the activity of AChE and results in no elongation of DP by TdT. Moreover, the control experiment, through substituting dNTP pool for dATP + dGTP pool, was also carried out to confirm that the signal change comes from the G-quadruplex generated by TdT. As expected, Figure S4 just exhibited a slight signal change in the dNTP pool reaction system duo to the randomly generated ssDNA. While obvious signal changes were observed in the dATP + dGTP reaction pool, showing that the luminescence enhancement of the system originated from the specific interaction of Din Ir(III)–1 with the TdT-generated G-quadruplex. Taken together, these results unambiguously confirmed the detection principle illustrated in Scheme 1, indicating that the TdT-assisted signal transduction and amplification detection platform can sensitively assay pesticides.

Figure 3. A. The luminescent responses of Din Ir(III)–1 towards different conditions. (a) Din Ir(III)–1, (b) a + DP/Hg2+ + ATCh, (c) b + AChE, (d) c + dATP + dGTP + TdT and (e) d + aldicarb. B. Nondenaturing PAGE imaging of TdT-assisted label-free luminescent detection


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products: lane a, DP; lane b, DP/Hg2+; lane c, b + dATP + dGTP + TdT; lane d, c + AChE + ATCh; lane e, d + aldicarb. The concentrations of DP/Hg2+, ATCh, AChE, TdT and aldicarb were 1 µM, 30 nM, 1.0 mU/mL, 30 U/mL and 200 µg/L, respectively. C.

Several experimental factors that may affect the performance of this analysis were optimized. In the optimization experiments, the relative luminescence intensities were tested in the absence of pesticide because of the “switch-off” property of the detection platform. Firstly, AChEcatalyzed hydrolysis takes a vital role in this strategy, so its concentration and reaction time were investigated. Figure S5 indicates the influence of AChE on the luminescence responses of this assay. Obviously, the luminescence intensities increased positively as the concentrations of AChE grew to 1.0 mU/mL, but hardly varied when the concentration further rose to 2.0 mU/mL, showing that the optimal level of AChE should be 1.0 mU/mL. Figure S6 indicates the influence from AChE reaction time on this detection. Evidently, the luminescence intensities rose with the increase of AChE-catalyzed hydrolysis time up to 100 min, and further prolonging the reaction time to 125 min resulted in no obvious varying in the luminescence intensity. Secondly, the influence of its concentration on the luminescent responses of this detection system was also studied since ATCh acts as the reaction substrate of AChE. Figure S7 showed that the signal intensity increased gradually with the increase of ATCh amount and then reaches a plateau at 30 nM of ATCh. Hence, 30 nM was chosen as the optimum concentration of ATCh. Nextly, the concentration and reaction time of TdT were also optimized since they influence the generation amount of G-quadruplex in the detection system. Figure S8 showed that luminescence intensity increased evidently as the concentration of TdT increased from 0 to 30 U/mL. To obtain high sensitivity for this detection platform, sufficient amount of Din Ir(III)–1 is needed to produce high luminescent signal. However, too high concentration of Din Ir(III)–1 could result in high


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background signal. Thus, the influence of Din Ir(III)–1 concentration was lastly investigated by analyzing the luminescent enhancement of the detection system, which is the difference between the luminescence intensity of Din Ir(III)–1 in the presence of DP/Hg2+, ATCh, AChE, dATP, dGTP and TdT with buffered solution just in the presence of Din Ir(III)–1. As displayed in Figure S9, signal enhancement exhibited a fast increase as the Din Ir(III)–1 concentration increased from 1 µM to 2 µM, while the luminescent signal just showed a slight decrease when the Din Ir(III)–1 concentration was further increased to 2.5 µM. After optimization of the detection factors, the luminescent response of the system towards different concentrations of aldicarb (0 to 100 µg/L) was studied. In the absence of aldicarb, the detection system demonstrated a ca. 10-fold enhancement in the luminescence intensity (Figure 4A), ensuring a high signal-to-noise ratio. The luminescence intensities gradually decreased with the increase of aldicarb concentrations. The luminescence intensity demonstrated a linear relation y = –15.39x + 383.2 (R2 = 0.994) with aldicarb concentrations from 0.5 to 25 µg/L (Figure 4B). According to this linear relation, the detection limit of (LOD) this assay for aldicarb was estimated to be 0.37 µg/L (S/N=3), which is comparable or superior to those of previously reported luminescent pesticides assays (Table S3). Therefore, the developed luminescent Gquadruplex-selective probe Din Ir(III)–1 can be successfully used in label-free detection of pesticides.


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Figure 4. A. Luminescent spectra of Din Ir(III)–1 in response to various concentrations of aldicarb: 0, 0.5, 2, 5, 10, 15, 25, 50 and 100 µg/L. B. The relationship between luminescence intensity at λ = 575 nm and aldicarb concentration. Inset: linear plot of the change in luminescence intensity at λ = 575 nm versus aldicarb concentration. Error bars represent the standard deviations of the results from three independent experiments. Considering the principle of this detection strategy, this method is expected to be suitable for detection of other kinds of organophosphate and carbamate pesticides, which possess the ability to inhibit the activity of AChE. In order to confirm the versatility of this detection method, the luminescent intensities of this assay system in the presence of other frequently used pesticides, such as glyphosate, dibrom, parathion, methomyl and carbaryl, was also investigated, respectively. Figure S10 showed that the control sample presents a very high luminescent signal in the absence of pesticides. In contrast, significantly reduced emission signals were found when these pesticides were respectively added into the system. These results verify that this label-free luminescent detection system is also suitable for the analysis of other pesticides, displaying good versatility of the developed detection method. The practical application of this detection system in food sample assay was investigated by detecting the aldicarb in ginger samples. The evaluation experiment was carried out by spiking the prepared samples with 1, 5, 10, 15, 25, 50 and 100 µg/L of aldicarb, respectively. Figures 5A


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and B showed that the luminescent intensity decreased with the increased aldicarb concentration and has a linearly decrease with aldicarb in the concentration range of 1–25 µg/L. To further demonstrate the potential usage of this detection in real environmental monitoring, the lake water samples were performed a recovery experiment. The recovery experiment was carried out by spiking the water samples with different concentrations of aldicarb since the original samples contain little aldicarb, below the LOD of this assay. The analyzed result was listed in Table S4. The proposed strategy exhibited good recoveries ranging from 89.0% to 106.2% for aldicarb (1, 2, 5, 10, 15 and 25 µg/L) assay in spiked lake water samples. In order to confirm the practical application of the proposed strategy in aldicarb detection, the levels of aldicarb in the lake water samples were also analyzed through the typical HPLC technique according to the reported methods.

32,61

The HPLC results were in accord with those data tested by the proposed strategy

(Figure S11), manifesting a reliable applicability. Therefore, the developed luminescent Gquadruplex-selective probe Din Ir(III)–1 can also be successfully used in real sample detection.

Figure 5. A. Luminescent spectra of Din Ir(III)–1 in response to various concentrations of aldicarb: 0, 0.5, 2, 5, 10, 15, 25, 50 and 100 µg/L in ginger extract. B. The relationship between luminescence intensity at λ = 575 nm and aldicarb concentration in ginger extract. Error bars represent the standard deviations of the results from three independent experiments. CONCLUSION


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In summary, the novel luminescent dinuclear Ir(III) complex Din Ir(III)–1 was designed and synthesized as a specific sensor to demonstrate excellent selective recognition towards Gquadruplex DNA over dsDNA or ssDNA and further to establish a label-free G-quadruplexbased detection platform for sensitive “signal-off” pesticides assay. The detection platform combines AChE-catalyzed reaction-mediated DNA/Hg2+ conformational change and signal transduction and amplification via TdT-directed G-quadruplex formation. In the presence of pesticides, the activity of AChE was inhibited and thus it causes the non-generation of the substrate TCh, which can sequester Hg2+ from the hairpin DNA/Hg2+ hybrid. As a result, TdT cannot elongate this blunt-ended hairpin DNA/Hg2+ hybrid through generating G-quadruplex froming DNA sequence, leading to reduced luminescent signal. The calibration curve of aldicarb detection showed a linear range of 0.5–25 µg/L and a LOD as low as 0.37 µg/L was obtained. Moreover, satisfactory results were successfully achieved by employing this strategy to detect pesticides contained in ginger or lake water samples. Therefore, the developed Din Ir(III)–1 can be used in construction of label-free G-quadruplex-based detection platform for analysis of pesticides in food safety or environmental monitoring fields.

ASSOCIATED CONTENT Supporting information General experimental; Materials; Preparation of stock solutions; Photophysical measurement; G4-FID assay; Luminescence response of iridium(III) complex towards different forms of DNA; Loop effect experiment for Din Ir(III)−1; Detection of aldicarb in real samples; The DNA sequences used in this work; Photophysical properties of Din Ir(III)–1; Comparison of our strategy with other luminescent methods for pesticides assay; Determination of aldicarb spiked in lake water samples; Synthesis of the N^N ligand L–1; G4-FID titration curves of ds-17 and


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PS2.M; Luminescence responses of Din Ir(III)–1 to various G-quadruplex DNA; Luminescence responses of Din Ir(III)–1 to dNTP pool or dGTP-rich pool; Luminescence responses of Din Ir(III)–1 to various concentrations of AChE; Luminescence responses of Din Ir(III)–1 to various reaction time of AChE; Luminescence responses of Din Ir(III)–1 to various concentration of ATCh; Luminescence responses of Din Ir(III)–1 to various concentration of TdT; Luminescent responses of the system to various concentration of Ir(III)–1; Luminescent responses of the system to other proteins; Analytical results of aldicarb in lake water samples obtained by using this method or by using HPLC. These experimental data in this study is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author F. Li., Tel/Fax: 86-532-86080855, E-mail: [email protected] Author Contributions The manuscript includes contributions from all authors. All authors have approved the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

This work was funded by the National Natural Science Foundation of China (Nos. 21705089 and 21775082), the Natural Science Foundation of Shandong Province (No. ZR201702080032), the Project of Shandong Province Higher Educational Science and Technology Program


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(J17KA109) the Special Foundation for Distinguished Taishan Scholar of Shandong Province (No. ts201511052), the Research Foundation for Distinguished Scholars of Qingdao Agricultural University (No. 663-1116010) and the Major Basic Research Program of Natural Science Foundation of Shandong Province (No. ZR2018ZC0127).

REFERENCES (1) Liu, G.; Guo, W.; Yin, Z. Biosens. Bioelectron. 2014, 53, 440-448. (2) Aragay, G.; Pino, F.; Merkoçi, A. Chem. Rev. 2012, 112, 5317-5338. (3) Hua, X.; Yin, W.; Shi, H.; Li, M.; Wang, Y.; Wang, H.; Ye, Y.; Kim, H. J.; Gee, S. J.; Wang, M. Anal. Chem. 2014, 86, 8441-8447. (4) Miao, Y.; He, N.; Zhu, J. J. Chem. Rev. 2010, 110, 5216-5234. (5) Dzudzevic, C. H.; Soylemez, S.; Akpinar, Y.; Kesik, M.; Gã¶Ker, S.; Gunbas, G.; Volkan, M.; Toppare, L. Acs Appl. Mater. Interfaces 2016, 8, 8058-8067. (6) Lee, J.; Lee, H. K. Anal. Chem. 2011, 83, 6856-6861. (7) Brito, N. M.; Navickiene, S.; Polese, L.; Jardim, E. F.; Abakerli, R. B.; Ribeiro, M. L. J. Chromatogr. A 2002, 957, 201-209. (8) Qian, G.; Wang, L.; Wu, Y.; Zhang, Q.; Sun, Q.; Liu, Y.; Liu, F. Food Chem. 2009, 117, 364-370. (9) Liu, G.; Wang, J.; Barry, R.; Petersen, C.; Timchalk, C.; Gassman, P. L.; Lin, Y. Chemistry 2010, 14, 9951-9959. (10) Zhang, Y.; Arugula, M. A.; Kirsch, J. S.; Yang, X.; Olsen, E.; Simonian, A. L. Langmuir 2015, 31, 1462-1468. (11) Yao, G. H.; Liang, R. P.; Huang, C. F.; Wang, Y.; Qiu, J. D. Anal. Chem. 2013, 85, 1194411950. (12) Liu, X.; Song, M.; Hou, T.; Li, F. Acs Sens. 2017, 2, 562-569. (13) Arduini, F.; Guidone, S.; Amine, A.; Palleschi, G.; Moscone, D. Sensor Actuat. B-Chem. 2013, 179, 201-208. (14) Dutta, R. R.; Puzari, P. Biosens. Bioelectron. 2014, 52, 166-172. (15) Kandimalla, V. B.; Ju, H. Chemistry 2006, 12, 1074-1080. (16) Ge, X.; Tao, Y.; Zhang, A.; Lin, Y.; Du, D. Anal. Chem. 2013, 85, 9686-9691. (17) Zhang, L.; Zhang, A.; Du, D.; Lin, Y. Nanoscale 2012, 4, 4674-4679. (18) Chen, H.; Zhang, H.; Yuan, R.; Chen, S. Anal. Chem. 2017, 89, 2823. (19) Wang, B.; Wang, H.; Zhong, X.; Chai, Y.; Chen, S.; Yuan, R. Chem. Commun. 2016, 52, 5049-5052. (20) Liu, D.; Chen, W.; Wei, J.; Li, X.; Wang, Z.; Jiang, X. Anal. Chem. 2012, 84, 4185-4191. (21) Qian, S.; Lin, H. Anal. Chem. 2015, 87, 5395-5400. (22) Li, Y.; Bai, H.; Li, C.; Shi, G. Acs Appl. Mater. Interfaces 2011, 3, 1306-1310.


20

Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(23) Liang, M.; Fan, K.; Pan, Y.; Jiang, H.; Wang, F.; Yang, D.; Lu, D.; Feng, J.; Zhao, J.; Yang, L. Anal. Chem. 2013, 85, 308-312. (24) Saa, L.; Grinyte, R.; Sã, n.-I. A.; Liz-MarzÃ n, L. M.; Pavlov, V. Acs Appl. Mater. Interfaces 2016, 8, 11139-11146. (25) Fahimikashani, N.; Hormozinezhad, M. R. Anal. Chem. 2016, 88, 8099-8106. (26) Liao, S.; Han, W.; Ding, H.; Xie, D.; Tan, H.; Yang, S.; Wu, Z.; Shen, G.; Yu, R. Anal. Chem. 2013, 85, 4968-4973. (27) Mccarthy, M. M.; Thompson, A.; Rivers, S.; Jahanzeb, M. Angew. Chem. 2007, 119, 78827886. (28) Liao, D.; Chen, J.; Zhou, H.; Wang, Y.; Li, Y.; Yu, C. Anal. Chem. 2013, 85, 2667-2672. (29) Kang, T. W.; Jeon, S. J.; Kim, H. I.; Park, J. H.; Yim, D. B.; Lee, H. R.; Ju, J. M.; Kim, M. J.; Kim, J. H. Acs Nano 2016, 10, 5346-5353. (30) Liao, S.; Han, W.; Ding, H.; Xie, D.; Tan, H.; Yang, S.; Wu, Z.; Shen, G.; Yu, R.; Chem, A. Anal. Chem. 2013, 85, 4968-4973. (31) Tian, T.; Li, X.; Cui, J.; Li, J.; Lan, Y.; Wang, C.; Zhang, M.; Wang, H.; Li, G. Acs Appl. Mater. Interfaces 2014, 6, 15456-15462. (32) Yi, Y.; Zhu, G.; Liu, C.; Huang, Y.; Zhang, Y.; Li, H.; Zhao, J.; Yao, S.; Chem, A. Anal. Chem. 2013, 85, 11464-11470. (33) Zhang, C.; Wang, L.; Tu, Z.; Sun, X.; He, Q.; Lei, Z.; Xu, C.; Liu, Y.; Zhang, X.; Yang, J. Biosens. Bioelectron. 2014, 55, 216-219. (34) Xu, Y.; Li, H.; Han, X.; Su, X. Biosens. Bioelectron. 2015, 74, 277-283. (35) Li, W.; Li, W.; Hu, Y.; Xia, Y.; Shen, Q.; Nie, Z.; Huang, Y.; Yao, S. Biosens. Bioelectron. 2013, 47, 345-349. (36) Nsibande, S. A.; Forbes, P. B. Anal. Chim. Acta 2016, 945, 9-22. (37) Castor, K. J.; Metera, K. L.; Tefashe, U. M.; Serpell, C. J.; Mauzeroll, J.; Sleiman, H. F. Inorg. Chem. 2015, 54, 6958-6967. (38) Shi, H.; Sun, H.; Yang, H.; Liu, S.; Jenkins, G.; Feng, W.; Li, F.; Zhao, Q.; Liu, B.; Huang, W. Adv. Funct. Mater. 2013, 23, 3268-3276. (39) Shi, H.; Sun, H.; Yang, H.; Liu, S.; Jenkins, G.; Feng, W.; Li, F.; Zhao, Q.; Liu, B.; Huang, W. Adv. Funct. Mater. 2016, 26, 6505-6505. (40) Ma, D. L.; Zhang, Z.; Wang, M.; Lu, L.; Zhong, H. J.; Leung, C. H. Chem. Biol. 2015, 22, 812-828. (41) Lu, L.; Chan, S. H.; Kwong, D. W. J.; He, H. Z.; Leung, C. H.; Ma, D. L. Chem. Sci. 2014, 5, 4561-4568. (42) Leung, K. H.; He, H. Z.; Wang, W.; Zhong, H. J.; Chan, D. S.; Leung, C. H.; Ma, D. L. Acs Appl. Mater. Interfaces 2013, 5, 12249-12253. (43) Lu, L.; Wang, M.; Mao, Z.; Kang, T. S.; Chen, X. P.; Lu, J. J.; Leung, C. H.; Ma, D. L. Sci. Rep. 2016, 6, 22458. (44) Du, Y. C.; Zhu, Y. J.; Li, X. Y.; Kong, D. M. Chem. Commun. 2018, 54, 682-685.


21


Page 22 of 22

(45) Hu, Y.; Shen, Q.; Wang, L.; Liu, Z.; Zhou, N.; Yao, S. Biosens. Bioelectron. 2015, 63, 331338. (46) Wang, L. J.; Luo, M. L.; Zhang, Q.; Tang, B.; Zhang, C. Y. Chem. Commun. 2017, 53, 11016-11019. (47) Tjong, V.; Yu, H.; Hucknall, A.; Chilkoti, A. Anal. Chem. 2013, 85, 426-433. (48) Chow, D. C.; Lee, W. K.; Zauscher, S.; Chilkoti, A. J. Am. Chem. Soc. 2005, 127, 1412214129. (49) Shi, K.; Dou, B.; Yang, J.; Yuan, R.; Xiang, Y. Biosens. Bioelectron. 2016, 87, 495-502. (50) Liu, Z.; Li, W.; Nie, Z.; Peng, F.; Huang, Y.; Yao, S. Chem. Commun. 2014, 50, 6875-6878. (51) Leung, K. H.; He, B.; Yang, C.; Leung, C. H.; Wang, H. M.; Ma, D. L. Acs Appl. Mater. Interfaces 2015, 7, 24046-24052. (52) Guédin, A.; Alberti, P.; Mergny, J.-L. Nucleic Acids Res. 2009, 37, 5559−5567. (53) Balasubramanian, S.; Hurley, L. H.; Neidle, S. Nat. Rev. Drug Discovery 2011, 10, 261− 275. (54) Guédin, A.; Gros, J.; Alberti, P.; Mergny, J.-L. Nucleic Acids Res. 2010, 38, 7858-7868. (55) Collie, G. W.; Parkinson, G. N. Chem. Soc. Rev. 2011, 40, 5867-5892. (56) Kumar, S.; Sharma, T. R. Oriental J. Chem. 2012, 28, 963-967. (57) Zhao, Q.; Liu, S.; Shi, M.; Wang, C.; Yu, M.; Li, L.; Li, F.; Yi, T.; Huang, C. Inorg. Chem. 2006, 45, 6152-6160. (58) Lu, L.; Zhong, H. J.; Wang, M.; Ho, S. L.; Li, H. W.; Leung, C. H.; Ma, D. L. Sci. Rep. 2015, 5, 14619. (59) Lin, S.; Gao, W.; Tian, Z.; Yang, C.; Lu, L.; Mergny, J. L.; Leung, C. H.; Ma, D. L. Chem. Sci. 2015, 6, 4284-4290. (60) Lu, L.; Mao, Z.; Kang, T. S.; Leung, C. H.; Ma, D. L. Biosens. Bioelectron. 2016, 85, 300308. (61) Jin, D.; Xu, Q.; Yu, L.; Mao, A.; Hu, X. Food Chem. 2016, 194, 959-965.


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Development of a Luminescent Dinuclear Ir(III) Complex for

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