Silver Ion As a Novel Coreaction Accelerator for Remarkably

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Silver Ion As a Novel Coreaction Accelerator for Remarkably Enhanced Electrochemiluminescence of PTCA/S2O82- System and Its Application in Ultrasensitive Assay of Mercury Ion Yan-Mei Lei, Rui-Xin Wen, Jia Zhou, Ya-Qin Chai, Ruo Yuan, and Ying Zhuo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b01018 • Publication Date (Web): 18 Apr 2018 Downloaded from http://pubs.acs.org on April 18, 2018

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Analytical Chemistry

Silver Ion As a Novel Coreaction Accelerator for Remarkably Enhanced Electrochemiluminescence of PTCA/S2O82- System and Its Application in Ultrasensitive Assay of Mercury Ion Yan-Mei Lei, Rui-Xin Wen, Jia Zhou, Ya-Qin Chai, Ruo Yuan∗, Ying Zhuo∗

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715.

∗ Corresponding authors at: Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected] (R. Yuan)[email protected](Y.Zhuo);. 1

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Analytical Chemistry 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

ABSTRACT：： In this work, with the use of Ag (I) ion as a robust coreaction accelerator toward enhancement of 3,4,9,10-perylenetetracarboxylic acid/peroxydisulfate (PTCA/S2O82-) system, a highly sensitive solid-state electrochemiluminescence (ECL) biosensing platform was successfully designed for mercury ion (Hg2+) detection. Specifically, a long guanine-rich (C-rich) double-stranded DNA (dsDNA) was generated by the target Hg2+-controlled DNA machine, which could embed the Ag (I) ion for ECL signal amplification of PTCA/S2O82- system. Herein, the Ag (I) ion as a coreaction accelerator, could first react with S2O82- to produce the Ag (II) ion and sulfate radical anion (SO4•−). Then, the companying Ag (II) ion could react with H2O to generate the reactive intermediate species (i.e. hydroxyl radical (OH•)), which could further accelerate the reduction of S2O82- to output more SO4•−. Moreover, the recycle of Ag (I) ion and Ag (II) ion was easily achieved by the electrochemical reaction. Therefore, an analanche-type reaction was triggered to generate massive amounts of SO4•−, which could react with the luminophore (PTCA) to achieve an extremely strong ECL signal. The ECL mechanism was investigated by ECL, cycle voltammetry (CV), fluorescence (FL) spectrum, ECL spectrum, and electron paramagnetic resonance (EPR) spectrum. As a result, the proposed solid-state ECL biosensing platform for Hg2+ detection exhibited high sensitivity with a linear range from 1×10-15 M to 1×10-10 M with a detection limit of 3.3×10-16 M. Importantly, this work firstly utilized metal ion as coreaction accelerator and provided a promising approach to improve the sensitivity of target analysis in ECL biosensing fields.

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KEYWORDS: Electrochemiluminescence, 3,4,9,10-perylenetetracarboxylic acid, coreaction accelerator, peroxydisulfate, mercury ion.

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1 Introduction It is generally known that the strong electrochemiluminescence (ECL) intensity is an essential prerequisite to preserve the high sensitivity for trace target detection in biosensing fields. Until now, great advances have been made in the improvement of ECL intensity toward binary (luminophore/coreactant) system. For example, the use of efficient coreactant or the intermolecular coreaction could availably speed up the electrochemical reaction rate (ERR) of luminophore and coreactant, which could further enhance the ECL intensity.1,2 Despite its exclusive popularity, the implementation of these approaches was fundamentally restricted by the dependence on the high concentration of luminophore and coreactant. Fortunately, our research group have recently found another promising approach, namely coreaction accelerator amplification strategy, where the coreaction accelerator was introduced in ECL binary system to improve the electrochemical reactivity of coreactant.3 Specifically, the coreaction accelerator could interact with coreactant instead of luminophore to generate more reactive intermediate states to improve the ERR of luminophore and coreactant, achieving a tremendous amount of the excited state of luminophore and emitting a strong ECL signal. Considering that the coreaction accelerator indeed improved the ECL intensity of binary system though the interaction with coreactant, it is significant to improve the utilization efficiency of coreaction accelerator and investigate the interaction mechanism in ternary system.4,5 Peroxydisulfate (S2O82-), as a classical coreactant,6,7 possessed the fascinating characteristics of simplicity, availability, and cost effectiveness, which could be

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electrochemically reduced to a strongly oxidizing intermediate of sulfate radical anion (SO4•−) and improve the ECL intensity of various luminophores, such as metal complexes (ie. Ru(bpy)32+), semiconductor nanoparticles (ie. CdSe quantum dot), and polyaromatic hydrocarbons (ie. rubrene).8-10 However, the high concentration of S2O82- could inevitably cause damage to the biosensing interface owing to its strong oxidizing property. Based on this, in our previous work, the ECL ternary system was obtained by introduction of the coreaction accelerator, such as amine compounds (ie. semicarbazide), metal oxide (Fe3O4-CeO2) and metal complexes (ie. hemin), which could react with S2O82- at a relatively low concentration to generate more SO4•− and accelerate the ERR of luminophore and S2O82- to amplify the ECL signal response.3,11,12 Consequently, it is highly desired to look for a robust coreaction accelerator with high-efficiency and regeneration, which has more promising applications in ECL biosensing fields. Herein, we first utilized the metal ion, Ag (I) ion, as coreaction accelerator in PTCA/S2O82- system to remarkably enhance the ECL intensity. Specifically, the Ag (I) ion could react with S2O82- to obtain the oxidant intermediates of sulfate radical anion (SO4•−) and Ag (II) ion, and then the companying Ag (II) ion could react with H2O to produce the more reactive intermediate species (ie. hydroxyl radicals (OH•)), which could further accelerate the reduction of S2O82- to generate more SO4•−. Moreover, the recycle of Ag (I) ion and Ag (II) ion was easily achieved by the electrochemical reaction. Therefore, an analanche-type reaction was triggered to generate massive amounts of SO4•−, which could react with the luminophore (PTCA) to produce more excited state of

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luminophore (1PTCA*), achieving an extremely strong ECL signal. Inspired by these advantages, in this work, a highly sensitive solid-state ECL biosensing platform was designed for mercury ion (Hg2+) detection (as shown in Scheme 1) based on Ag (I) ion as a robust coreaction accelerator for the signal enhancement of PTCA/S2O82- system. First, the PTCA supramolecular nanorods were obtained by a simple solvent-vapor-driven self-assembly method, which not only acted as a substrate material possessing desirable electrical conductivity, excellent membrane forming ability and large specific surface area, but also acted as a excellent luminescent material showing ECL emission in S2O82− solution. Then Au nanopaticles were electrochemically deposited on the above electrode surface for hairpin probe H1 (with a thiol at the 5’-end) loading. In the presence of target Hg2+, the simple and flexible DNA machine was triggered to form the “duplicator-like” DNA machine via thymine-Hg2+-thymine interaction, which further initiated the strand-displacement amplification (SDA) reaction with the assistance of polymerase and nicking endonuclease (Nt.BbvCI), resulting in the generation of massive mimic target (MT). Subsequently, the MT was captured by the hairpin probe 1 (H1) and further triggered the hybridization chain reaction (HCR) to generate the long guanine-rich (C-rich) dsDNA in the presence of hairpin probes (the hairpin probes 1 and 2 were abbreviated as H2 and H3). After incubating with AgNO3 solution, Ag(I) was captured by the C-rich dsDNA via C-C mismatch to form the stable Ag(I) embedded DNA complexes (DNA-Ag(I)). With the use of Ag (I) ion as an efficient coreaction accelerator, the solid-state ECL biosensing platform achieved highly

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sensitivity for Hg2+ detection. The use of metal ion as coreaction accelerator opened a new way to improve the sensitivity of target analysis in ECL biosensing fields.

2 Experimental section 2.1 Preparation of 3,4,9,10-perylenetetracarboxylic Acid Suspension Solution. All instruments, reagents and DNA sequences used in this work as shown in the Supporting Information 1.1 and 1.2.

The 3,4,9,10-perylenetetracarboxylic acid (PTCA) suspension solution was prepared as follows: First, the PTCA was synthesized from hydrolyzed perylene-3,4,9,10-tetracarboxylic dianhydride (PTCDA) by a modified Caruso method.13 After that, the above mixture was centrifuged and washed with ultrapure water until the pH was 7.4, and then dried at 60 oC for 12 h. Subsequently, 5.0 mg PTCA powder was dissolved in 5.0 mL acetonitrile solution with vigorous stirring under room temperature, achieving a homogeneous, red suspension solution. The resultant PTCA suspension solution was stored in the refrigerator at 4 oC when not in use. 2.2 Fabrication of the Solid-state ECL Biosensing Platform. Prior to use, the glassy carbon electrode (GCE, Φ = 4.0 mm) was polished and cleaned according to the method reported previously.3 First, 5 µL PTCA suspension was dropped onto the clean GCE surface to in situ form PTCA supramolecular nanorods at room temperature via a simple solvent-vapor-driven self-assembly

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method.14 Following that, the PTCA modified electrode was immersed in 2 mL HAuCl4 solution (1%) to achieve AuNPs under a constant potential for 30 s at −0.2 V. Next, 10 µL H1 solution (0.5 µM) in the buffer solution (10 mM Tris-HCl, 10 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP), 0.1 M NaCl, pH 7.4) was dropped on the above modified electrode and incubated overnight at room temperature. Finally, the resultant modified electrode (GCE/PTCA/AuNPs/H1) was incubated with 15 µL hexanethiol (HT, 1 mM) for 40 min to block the nonspecific binding sites. After each step, the modified electrode was thoroughly cleaned with ultrapure water to remove the physically absorbed species. 2.3 Measurement Procedure. First of all, the target Hg2+ was converted into mimic target (MT) as follows. First, the different concentrations of Hg2+ sample solution and 5 µM machine DNA in 100 µL 1 × NEB buffer solution was heated to 37 oC for 1 h. Then, the above mixture containing phi29 DNA polymerase (0.05 U/µL), Nt.BbvCI (1 U/µL), and 500 µM deoxynucleotide triphosphates (dNTPs) was performed at 37 oC for 2 h, achieving massive MT due to the strand displacement amplification (SDA). Finally, the above reaction was stopped by a thermal treatment at 80 oC for 10 min, and then allowed to cool down to room temperature.

The measurement procedure was performed as the following steps. First, the proposed ECL biosensor was incubated with 10 µL MT solution for 2 h at room temperature. After that, the above modified electrode was further incubated with 20

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µL the mixture of H1 and H2 (0.5 µM) for 2 h to trigger the HCR amplification with the generation of the long C-rich dsDNA. Next, 10 µL Ag NO3 solution (1 mM) was dropped on the above modified electrode surface for 60 min to obtain the finished biosensor (GCE/PTCA/AuNPs/H1/MT/DNA-Ag(I)). After each step, the modified electrode was thoroughly cleaned with ultrapure water to remove the physically absorbed species. Finally, the ECL measurement was detected by a MPI-E ECL analyzer in 3 mL K2S2O8 (5 mM, pH 7.4) solution.

Scheme 1. Schematic Illustration of the Preparation and Detection Process of the ECL Biosensor for Target Hg2+ Detection: (A) The Target Hg2+-controlled Actuation of DNA Machine to Induce SDA Reaction. The Possible Mechanism of PTCA/S2O82- Without (B) and With (C) the 9



Coreaction Accelerator of Silver Ions.

3 Results and discussion 3.1. Characterization of the Different Nanomaterials The sizes and morphologies of the as-prepared nanomaterials were characterized by scanning electron microscopy (SEM). According to Figure 1A, the PTCA obviously showed short rod-like structures with diameter of around 250 nm and length of up to about 1 µm, which derived from the π~π stacking and hydrogen bonding similar to previous reports.15,16 In addition, the sea cucumber-like PTCA/AuNPs were displayed in Figure 1B. It could be clearly observed that large amounts of the approximately spherical Au NPs were well-distributed on the PTCA nanorod surface. X-ray photoelectron spectrometry (XPS) was used to investigate the proposed interface after generation of DNA-Ag (I), as shown in Figure 1 C and 1D. The Au 4f, C 1s, N 1s, Ag 3d, and O 1s peaks appeared in the XPS survey spectrum, whereas the Ag 3d peaks indicated that the Ag (I) ion was successfully intercalated into the dsDNA grooves via C-C mismatch.

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Figure 1. SEM images of (A) short rod-like PTCA and (B) sea cucumber-like PTCA/AuNPs, respectively. (C) XPS analysis for full region of the finished biosensor. (D) The different elements of C1s region, O1s region, Ag3d region, Au4f region, and N1 region.

3.2. Feasibility Investigation of the Biosensor for Hg2+ Detection. In order to confirm the feasibility analysis of the biosensor for Hg2+ detection based on Hg2+-triggered SDA to drive HCR amplification, the gel electrophoresis was employed to characterize the different samples. As shown in Figure 2, the mixed solution of target Hg2+, DNA machine, phi29 polymerase, Nt.BbvCI, and dNTPs was shown in lane 1. Two bands consisted of a distinct band, corresponding to the DNA machine, and another unconspicuous band, corresponding to the MT (single strand DNA produced from SDA).17 However, only a distinct single band was observed in above mixture without target Hg2+ (Lane 2), indicating that no SDA reaction occurs 11



because of no MT band appeared. Subsequently, the mixture of H1, H2, and H3 was shown in lane 3. A remarkable single band was observed at the bottom owing to the identical base number of H1, H2, and H3, indicating that no HCR occurs. After the addition of MT solution in the mixture of H1, H2, and H3, an obvious band was observed at the top with much slower migration (Lane 4), indicating that MT could trigger HCR to form a long dsDNA.18 As expected, these results indicated that target Hg2+ could trigger SDA to drive HCR amplification.

Figure 2. PAGE analysis of different samples. Lane 1: MT solution (the mixture of 2 µM DNA machine + 0.05 U/µL phi29 polymerase + 1 U/µL Nt.BbvCI + 500 µM dNTPs + 1 fM Hg2+ was executed by the proposed method shown in experimental section 2.3); Lane 2: MT solution without Hg2+; Lane 3: 2 µM H1 + 2 µM H2 + 2 µM H3; Lane 4: HCR product (2 µM H1 + 2 µM H2 + 2 µM H3 + MT solution).

3.3. The Possible ECL Mechanism of the Different System.

To investigate the possible ECL mechanism of Ag (I) ion towards PTCA/S2O82system, the ECL potential profiles of the different systems were obtained with the cyclic potential scanning between −1.7 V and 0 V, and these results were shown in

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Figure 3A. The curve a exhibited an ECL peak (about 1251 a.u) of bare GCE in 5 mM S2O82- solution (pH 7.4) correponding to the emission of singlet oxygen (1(O2)2*).3 Subsequently, when the modified electrode GCE/PTCA was measured in S2O82- solution, an evidently ECL peak intensity about 4416 a.u (curve b) was observed corresponding to the emission of the excited state of PTCA (1PTCA*).19 Remarkably, when the finished biosensor (GCE/PTCA/AuNPs/H1/MT/DNA-Ag(I)) was detected in S2O82- solution, the ECL intensity was significantly increased by 1.72 times (12032 a.u, curve c) in comparison with that of curve b. The reason for this was that the Ag (I) ion as a robust coreaction accelerator could effectively improve the ERR of PTCA and S2O82- to amplify ECL response. Additionally, as shown in the inset of curve a in Figure 3A, the maximum ECL peak position was appeared earlier in presence of Ag (I) ion in comparison with that of curve b (without Ag (I) ion), further indicating that Ag (I) ion could effectively improve the ERR of the PTCA and S2O82-. More interestingly, when the finished biosensor without PTCA was measured in the S2O82- solution, the ECL signal about 2316 a.u was evidently increased (curve d ) by 1.91 times in comparison with that of curve a, demonstrating that the Ag (I) ion could promote the reduction of S2O82- to produce more SO4•−, and thus resulting in the stronger emission of 1(O2)2*. However, when the finished biosensor was detected in 0.1 M PBS solution (pH 7.4), predictably, almost no ECL signal was observed in the same potential range (curve e), demonstrating that the Ag (I) ion could not interact with PTCA to amplify the ECL signal.

To further clarify the ECL mechanism of Ag (I) ion in the PTCA/S2O82- system, 13



the CV curves of bare GCE and the finished biosensor were obtained in S2O82solution, respectively (as shown in Figure 3B). The bare GCE in S2O82- solution exhibited an obvious reduction peak at −0.98 V (curve a), which indicated that the strong oxidant of SO4•− was generated by electrochemical reduction of S2O82-.20 However, the CV curves of the finished biosensor showed the corresponding reduction peak potential shifted positively (−0.71 V) and peak current increased distinctly (curve b) in comparison that of curve a, which demonstrated that the silver (I) ions could accelerate the interaction between PTCA and S2O82-.

Figure 3. (A) ECL-potential profiles: (a) bare GCE, (b) GCE/PTCA, and (c) the finished biosensor (GCE/PTCA/AuNPs/H1/MT/DNA-Ag(I)) in 5 mM S2O82- solution, respectively. And (d) the finished biosensor in 0.1 M PBS solution, (e) the finished biosensor without PTCA in 5 mM S2O82- solution. The inset of (A): ECL-time profiles of curve b and curve c. (scan range: −1.7

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to 0 V, vs Ag/AgCl). (B) CV responses of (a) bare GCE and (b) the finished biosensor in 5 mM S2O82- solution. (C) Normalized ECL spectrum of (a) bare GCE, (b) the finished biosensor without PTCA, (c) GCE/PTCA, and (d) the finished biosensor in 5 mM S2O82- solution (scan range: −1.7 to 0 V) were obtained by the optical filter, respectively. (D) Fluorescence spectrum of PTCA solution (pH 10.0): (a) excitation spectrum and (b) emission spectrum.

The ECL spectrum were also obtained to further identify the possible ECL mechanism of Ag (I) ion in the PTCA/S2O82- system, as shown in Figure 3C. The ECL spectrum of bare GCE (curve a) and the finished biosensor without PTCA (curve b ) in S2O82- solution showed the emission peak at 575 nm, confirming that the luminophore was the 1(O2)2*.3 Remarkably, the ECL spectrum of GCE/PTCA (curve c) and the finished biosensor (curve d ) in S2O82- solution exhibited the emission peak at 492 nm, which matched with the FL spectrum of PTCA (Figure 3D, curve b). These results further demonstrated that the luminophore was the 1(PTCA)* and the Ag (I) ion acted as a robust coreaction accelerator in PTCA/S2O82- system. Consequently, the possible ECL mechanism of different systems: PTCA/PBS, PTCA/S2O82-, PTCA/S2O82-/Ag (I) ion, PTCA/Ag (I) ion, S2O82-, S2O82-/Ag (I) ion, could be depicted in the Supporting Information 1.3 (a ~ f ).

3.4. The Possible ECL Enhanced Effect and Mechanism of Ag (I) Ion Towards PTCA/S2O82- System.

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Figure 4. (A) Effects of the different coreaction accelerators on the ECL intensity for PTCA/S2O82- system in 5 mM S2O82- solution: (a) aniline, (b) hemin, and (c) Ag (I) ion solution. (B) EPR spectrum of (a) PBS + DMPO solution, (b) S2O82- + DMPO solution, (c) Ag (I) ion + DMPO solution, and (d) Ag (I) ion + S2O82- + DMPO solution. “●” represents DMPO-OH adducts; “×” represents DMPO-SO4 adducts.

To evaluate the ECL enhanced effect of Ag (I) ion toward PTCA/S2O82- system, we compared the ECL responses of GCE/PTCA in S2O82- solution containing the different coreaction accelerators of (a) aniline, (b) hemin, and (c) Ag (I) ion, respectively. As shown in Figure 4A, the ECL intensity increased with the increasing of coreaction accelerator concentration from 0 to 50 µM. The linear regression equation of PTCA/S2O82-/aniline system (curve a), PTCA/S2O82-/hemin system (curve b), and PTCA/S2O82-/Ag (I) ion system (curve c) were obtained as Ia = 60.40 lg ca+ 4349.42, Ib = 98.64 lg cb+ 4882.95, and Ic = 158.17 lg cc+ 5887.14, respectively (where I represents the ECL intensity and c represents the concentration of coreaction accelerator). As a results, the ECL slope rate of curve c, kc (kc = 158.17, with Ag (I) ion as coreaction accelerator) was higher than those of curve a (ka = 60.40, with aniline as coreaction accelerator) and curve b (kb = 98.64, with hemin as coreaction accelerator), which demonstrated that the Ag (I) ion as a coreaction

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accelerator had high efficiency in PTCA/S2O82- system.

To further clarify the ECL enhanced mechanism of Ag (I) ion towards PTCA/S2O82- system, the in situ electron paramagnetic resonance (EPR) spectrum with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) spin trap (as shown in Figure 4B) was conducted to detect and identify the radical species generation in the different systems. There were four different systems of (a) PBS + DMPO solution, (b) S2O82- + DMPO solution, (c) Ag (I) ion + DMPO solution, and (d) Ag (I) ion + S2O82- + DMPO solution, respectively. Remarkably, the hyperfine splitting constants aN = 13.8 G, aHβ = 10.2 G, and aHγ = 1.4 G were characteristic of the DMPO-SO4 adducts. Meanwhile, the characteristic EPR spectra consisted of 1:2:2:1 quartet pattern with hyperfine splitting constants aN ≈ aHβ ≈ 14. 9 G, which were representative of DMPO-OH adducts.21,22 First, no signals were observed in PBS + DMPO solution (curve a) and Ag (I) ion + DMPO solution (curve c), respectively, indicating that the neither PBS nor Ag (I) ion solution could produce OH• or SO4•−. Subsequently, a typical four-line EPR spectra with a peak intensity ratio of 1:2:2:1 were observed in S2O82- + DMPO solution (curve b), corresponding to the generation of more DMPO-OH products. However, the DMPO-SO4 signal was very weak, which derived from the reaction of SO4•− with H2O to form OH• and the self-scavenging effects of SO4•−.23 Interestingly, when Ag (I) ion added into S2O82- + DMPO solution, the DMPO-SO4 signal and DMPO-OH signal were significantly enhanced (curve d ), which indicated that the Ag (I) ion as a coreaction accelerator could efficiently interact with S2O82- to produce massive OH• and SO4•−. The reason may be that the 17



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Ag (I) ion could interact with S2O82- to form Ag(II) ions and SO4•−, and then the Ag (II) ion could further react with H2O to produce the more reactive intermediate species(ie, OH•), where the generated intermediate species could accelerate the reduction of S2O82- to form more SO4•− and the regeneration of Ag (I) ion.24,25

As discussed above, we could speculate that the Ag (I) ion as a high-efficiency coreaction accelerator toward the PTCA/S2O82- system arose from the massive SO4•− generated as the following two routes: (i) The Ag (I) ion could react with the S2O82to produce more SO4•− and Ag (II) ion (route I, eqs 1), and then the Ag (II) ion could further react with H2O to produce the more reactive intermediate species (ie. OH•), which could accelerate the reduction of S2O82- to generate more SO4•− and the regeneration of Ag (I) ion (route II, Eqs 2 - 6). Remarkably, the recycle of Ag (I) ion and Ag (II) ion was easily achievable with the electrochemical process. Therefore, an analanche-type reaction was triggered to generate massive amounts of SO4•−, which could react with the luminophore (PTCA) to produce the more excited state of luminophore (1PTCA*), achieving an extremely strong ECL signal. In addition, the possible ECL enhanced mechanism of the Ag (I) ion in PTCA/S2O82- system could be outlined in the following Eqs 1 - 9: Route I: Route II:

Ag(I) + S2O82− → Ag(II) + SO42− + SO4−• Ag(II) + H2O → Ag(I) + AgO++2H+

(1) (2)

AgO+ + H2O → Ag(I) + H2O2

(3)

H2O2 + e− → OH− + OH•

(4)

OH• + S2O82− → SO4−• + HSO4− + O2

(5)

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O2 + H2O + e− → OH− + H2O2

(6)

ECL emission： PTCA + e− → PTCA−•

(7)

PTCA−• + SO4−• → 1PTCA* + SO42−

(8)

1

PTCA* → PTCA + hv

(9)

3.5. Analytical Performance of the Solid-state ECL Biosensing Platform.

Figure 5. Sensitivity investigation of the prepared biosensor for Hg2+ detection. (A) ECL intensity-time curves of the prepared biosensor tested with different concentrations of Hg2+. (B) The corresponding calibration plot of the ECL intensity vs. the logarithm of Hg2+ concentration. (C) Selectivity of the prepared biosensor toward Hg2+ by comparing it to interfering metal ions. (D) Stability of the prepared biosensor toward 1×10−15 M Hg2+ under a continuous cyclic potential scan.

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To evaluate the analytical performance of the prepared biosensor, the prepared biosensor was incubated with different concentrations of Hg2+ and implemented the measurement procedure (as shown in experimental section 2.3). As shown in Figure 5A, under the optimal conditions, it could be seen that the ECL response increased accordingly with the increasing of Hg2+ concentration. The calibration curve exhibited a good linear relationship between ECL responses and the logarithmic values of Hg2+ concentrations from 1.0×10−15 M to 1.0×10−10 M (Figure 5B). The linear regression equation was expressed as I = 1136.03 lg c+ 20197.19 (where I stands for the ECL intensity and c stands for the concentration of Hg2+), with a correlation coefficient of 0.9978 and a detection limit of 3.3×10−16 M (S/N= 3). Additionally, the prepared biosensor was compared with other test platforms (Table 1), which indicated that the prepared biosensor was highly sensitive for Hg2+ detection. Table 1. Comparison of the Other Test Platforms for Hg2+ Detection. Methods

Detection Limit

Dynamic Range

Ref

fluorescence

4.5 × 10-12 M

1×10-11 M ~ 1×10-7 M

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electrochemical

2.8 × 10-11 M

1×10-10 M ~ 1×10-8 M

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resonance Rayleigh scattering

2.0 ×10-11 M

5×10-11 M ~ 5×10-7 M

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ECL

3.1 ×10-12 M

1×10-11 M ~ 1×10-3 M

29

ECL

2.0 ×10-15 M

5×10-14 M ~ 1×10-10 M

30

ECL

3.3 ×10-16 M

1×10-15 M ~ 1×10-10 M

This work

To further evaluate the selectivity and specificity of the prepared biosensor for detection of Hg2+, several interference metal ions, including Ca2+, K+, Cu2+, Li+, Mg2+, 20


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Ni2+, Pb2+, and Zn2+ at a concentration of 1.0 × 10-10 M, were tested under the same conditions as in the case of Hg2+ (1.0 × 10-12 M). As shown in Figure 5C, the interference metal ions exhibited negligible interference with Hg2+ selectivity owing to the specific T-Hg2+-T coordination. These results suggested that the prepared biosensor displayed a remarkably favorable selectivity and specificity for the determination of Hg2+ over interference metal ions. In addition, the stability of the prepared biosensor for Hg2+ were also investigated during consecutive cyclic potential scan about 12 cycles for the determination of Hg2+ (1.0 × 10-15 M). As shown in Figure 5D, it could be seen that the relative standard deviations (RSD) was 1.35%, indicating that the stability of proposed ECL biosensor was acceptable.

3.6 Direct Detection of Hg2+ in Real Samples.

To evaluate the applicability and reliability of the prepared biosensor for Hg2+ detection in real sample solution, the concentrations of Hg2+ in extracted soil solution (the soil solution was obtained as shown in the Supporting Information 1.5) were determined by proposed method as well as inductively coupled plasma mass spectrometric (ICP-MS) method. Considering of the bioavailability of Hg2+ in soil, the Hg2+ concentration of four sample solutions (exchangeable, carbonate, Fe-Mn oxides, and organic fractions) were successively detected as shown in Table 2. According to the statistic calculation by a t test (a = 0.1), the concentrations of the proposed method displayed fairly good concordance compared with that of ICP-MS method (t1 = 0.70, t2 = 0.43, t3 = 0.77 and t4 = 0.57, which were smaller than the

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standard t value of 2.13), indicating that the proposed method was accurate and reliable for Hg2+ detection in practical samples. Table 2. Determination of Hg2+ (fM) in Soil Extraction Solutions Using the Proposed Method and ICP-MS Method.

α

Sample

The proposed method

ICP-MS method

t value

exchangeable carbonate Fe-Mn oxides

1.54 ± 0.10 1.45 ± 0.11 0.74 ± 0.09

1.48 ± 0.11 1.41 ± 0.12 0.67 ± 0.13

0.70 0.43 0.77

organic fractions

0.52 ± 0.07

0.48 ± 0.10

0.57

Mean measured concentration of three replicates ± standard deviation.

4. Conclusions

In summary, the Ag (I) ion as an efficient coreaction accelerator toward enhancement of PTCA/S2O82- system was firstly utilized to construct the highly sensitive solid-state ECL biosensing platform for Hg2+ detection. Compared with the reported coreaction accelerators, the Ag (I) ion with high-utilization derived from the recycle of Ag (I) ion and Ag (II) ion, where an analanche-type reaction was triggered to generate massive amounts of the oxidant intermediates SO4•−. Thus, an extremely strong ECL signal was achieved due to the reaction of SO4•− and PTCA, producing a large number of 1PTCA*. In view of these advantages, the solid-state ECL biosensing platform achieved high sensitivity for Hg2+ detection. This strategy firstly utilized metal ion as coreaction accelerator and provided a promising approach to improve the sensitivity of target analysis in ECL biosensing fields.

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ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge via the Internet at http://pubs.acs.org/. Reagents

and

material,

apparatus,

schematic

illustration

of

possible

luminescence mechanism of different systems, ECL and CV characterization of the stepwise assembly of the ECL biosensor, pretreatment of the soil samples.

AUTHOR INFORMATION This work was financially supported by the NNSF of China (21675130, 21575116, 21675129, 21775124), and the Fundamental Research Funds for the Central Universities (XDJK2018AA003, XDJK2016E056), China.

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Silver Ion As a Novel Coreaction Accelerator for Remarkably

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