Electrochemiluminescent Pb2+-Driven Circular Etching Sensor

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Electrochemiluminescent Pb2+-Driven Circular Etching Sensor Coupled to a DNA Micronet-Carrier Wen-Bin Liang, Ying Zhuo, Ying-Ning Zheng, Cheng-Yi Xiong, Ya-Qin Chai, and Ruo Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12672 • Publication Date (Web): 20 Oct 2017 Downloaded from http://pubs.acs.org on October 23, 2017

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ACS Applied Materials & Interfaces

Electrochemiluminescent Pb2+-Driven Circular Etching Sensor Coupled to a DNA Micronet-Carrier Wen-Bin Liang,†, ‡ Ying Zhuo,† Ying-Ning Zheng,† Cheng-Yi Xiong,† Ya-Qin Chai,*,† and Ruo Yuan*,† †

Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University),

Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China ‡

Department of Clinical Biochemistry, Laboratory Sciences, Southwest Hospital, Third Military

Medical University, 30 Gaotanyan Street, Shapingba District, Chongqing 400038, PR China

*

Corresponding Author

E-mail: [email protected] (Yaqin Chai), [email protected] (Ruo Yuan). Tel.: +86-23-68252277; Fax: +86-23-68253172

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ACS Paragon Plus Environment


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ABSTRACT: Herein, an ultrasensitive electrochemiluminescent (ECL) strategy was designed based on the fabrication of a multi-interface DNA micronet-carrier via layer by layer hybridization of double-stranded DNAzyme-substrate to immobilize large amounts of ECL indicator, [Ru(dcbpy)2dppz]2+, in double-strand DNA on the electrode surface, generating enhanced ECL signals. When the double-stranded structures were cleaved circularly via Pb2+ in the detection sample, the ECL indicator was released, which resulted in a decreased ECL signal associated with the concentration of Pb2+, that had higher sensitivity and wider linear range. As a result, the developed ECL strategy exhibited a linear range from 50 pM to 500 µM with a detection limit of 4.73 pM, providing an alternative analytical strategy with excellent properties, including a high sensitivity and a wide linear range. Importantly, the ECL strategy could be readily expanded for various metal ions, proteins, nucleotide sequences and cells, offering a simple and efficient technology for both environmentally safe assays and clinical diagnostics. KEYWORDS: Electrochemiluminescent assay, Circular etching, Metal ion, Nucleotide sequences, DNA micronet, DNAzyme

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■INTRODUCTION Heavy metal ions, especially lead (II) ions (Pb2+), are major environmental pollutants that could harm numerous systems in the human body, including the neurological, hematological, gastrointestinal, cardiovascular and renal system, and these systems are especially vulnerable in children due to their immature nervous system.1-4 Developing a suitable detection technique to monitor the concentration of heavy metal ions in water for environmental safety and in blood and cells for clinical diagnostics has been urgently needed.5, 6 Electrochemiluminescent (ECL) assays have been shown to be powerful analytical techniques for the determination of various targets, including metal ions, proteins, nucleotide sequences and cells, especially with the development of nanotechnology and enzyme catalytic research.7-15 Lu and colleagues proposed a highly sensitive ECL assay for a Pb2+ assay based on the distance-dependent quenching effect of nanocomposites of graphene and gold nanoparticles on the ECL emission from CdSe quantum dots.11 Recently, we have constructed sensitive ECL assays by generating a Pb2+-based Gquadruplex structure. Significant advantages have been made in the sensitive detection of Pb2+.16, 17

Although the current approach is valuable, there are still challenges that need to be overcome

to develop an ultrasensitive strategy with a wider detection range for accurate monitoring of Pb2+ pollution. With the goal of improving detection sensitivity, various amplification strategies have been developed. One useful and widely applied strategy is the sandwich-type assay with detection elements labeled with signal probes, such as the detection antibodies or detection nucleotides. These strategies were difficult to use for the sensitive detection of heavy metal ions because the metal ions were too small to react with capture element and detection element simultaneously.

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The recent discovery of DNAzyme gave a new opportunity to generate suitable sensitive detection method for metal ions. Importantly, it is well known that various approaches have been used to improve detection sensitivity by amplifying the nucleotide sequence. Due to the high plasticity of DNA, it is relatively easy to design DNA with various functional structures. At the same time, numerous modifications could be easily introduced into these DNA structures. All of these properties make a DNA-based strategy with DNAzyme as an attractive approach for monitoring the concentration of Pb2+ efficiently.17-23 In recent years, various DNA-based strategies, especially DNAzyme-based strategies, have been reported for sensitive detection of Pb2+.18, 19 Lu's group reported a simple Pb2+ biosensor based on DNAzyme-directed assembly of gold nanoparticles that had high sensitivity and selectivity for Pb2+.19 Plaxco and colleagues proposed an electrochemical detection method for Pb2+ with good sensitivity and specificity via an electrode-bound DNAzyme assembly based on target-driven cleaving.22 It was mentioned that a recent reported amplification strategy via a target-driven circular cleaving strategy showed prefect performances for the ultrasensitive detection of various targets, which indicated great potential for ultrasensitive ECL detection of metal ions with the help of DNAzymes. In this study, an ECL strategy was designed based on the layer-by-layer fabrication of a DNA micronet-carrier with DNAzymes to immobilize ECL indicators on the electrode surface and Pb2+-driven circular etching on the DNA micronet-carrier to release ECL indicators, resulting in decreased ECL signals associated with the concentration of Pb2+ (Scheme 1). Typically, the electrode surface was modified first with a double-strand DNAzyme substrate layer-by-layer. The bis (4, 4'-dicarboxy-2, 2'-bipyridyl) (4, 5, 9, 14-tetraaza-benzo-[β]triphenylene) ruthenium(II) ([Ru(dcbpy)2dppz]2+, RU), which is one of the artificial ECL

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indicators, could be intercalated into double-strand DNA with high affinity, which could be released when the double-strand structure was cleaved specifically by Pb2+ based on the interaction between Pb2+ and DNAzyme substrate, which resulted significantly decreased ECL signal associated with the concentration of Pb2+.23-26 Furthermore, success in the establishment of DNAzyme-based strategy with the target-driven circular etching could be readily expanded for various metal ions, proteins, nucleotide sequences and cells, offering a simple and efficient technology for both environmentally safe assays and clinical diagnostics.

Scheme 1 Schematic diagram of the proposed ultrasensitive electrochemiluminescent strategy involving Pb2+-driven circular etching on the layered DNA micronet-carrier. ■EXPERIMENTAL METHODS Reagents and Materials. [Ru(dcbpy)2dppz]2+ was purchased from Suna Technology Inc. (Suzhou, China). Gold chloride (HAuCl4), lead nitrate (Pb(NO3)2) and trimethylamine (TPrA) were obtained from Sigma Chemical Co. (MO, USA). Phosphate buffered solutions (PBS, pH

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7.4) were prepared with 0.1 M K2HPO4, 0.1 M NaH2PO4, and 0.1 M KCl, and the pH was adjusted with 0.1 M HCl or NaOH. Dulbecco’s modified eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin were purchased from Gibco Laboratories Life Technologies Inc. (NY, USA). Ultrapure water (18.2 MΩ/cm) was prepared with a Millipore water purification system (MilliQ, Millipore Inc., MA, USA). All oligonucleotides were customsynthesized and purified from Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China). The nucleotide sequence information is listed in Table 1. Table 1. The oligonucleotides employed in the proposed electrochemiluminescent strategy. Name Capture Linker (CL)

Sequence (5'→3') TCG ATT CCC TGT (rA) GGG GGT TTT TT-SH

S1

TCC TTC CGA GCC GGT CGA AAT CCT TTT ACC AAC TA

S2

GAA TCG ATT ACC CCT CCG AGC CGG TCG AAA TCA GG

Assisted Linker (AL) Blocking DNA

TCG ATT CCC TGT (rA) GGG GGA GTA TTG CGG AGG AT (rA) G AGG ATA GTT CCT SH-TTT TTT

Apparatus. ECL measurements were performed on a model MPI-E II multifunctional electrochemical and chemiluminescent analytical system (Xi’an Remax Electronic Science and Technology Co., Xi’an, China) in which a conventional three-electrode system was employed with a Ag/AgCl (saturated KCl) as the reference electrode, a platinum wire as the auxiliary electrode, and a glassy carbon electrode (GCE) with/without modification as the working electrode, respectively. The electrochemistry measurements were carried out on a CHI 660D electrochemical workstation (Shanghai Chen Hua Instrument, Shanghai, China) with the same three-electrode system. Photophysical characterizations were performed with a RF-5301PC

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fluorescence

spectrophotometer

(Shimadzu

Co.,

Tokyo,

Japan)

and

a

UV-2550

spectrophotometer with standard quartz cuvettes. The ECL emission spectra were obtained on an electrochemical workstation combined with a Newton EMCCD spectroscopy detector (Andor Co., Tokyo, Japan). The morphology of the fabricated electrodes was evaluated out by dimension edge atomic force microscopy (AFM, Bruker Co., Germany). Cell Culture. The MDA-MB-231 human breast cancer cell line and A549 alveolar basal epithelial cell line were employed as a model to investigate the applicability of the proposed ECL assay, which were purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The MDA-MB-231 cells and A549 cells were maintained in a humidified atmosphere that contained 5 % CO2 at 37 °C and cultured in DMEM medium and F12 medium, respectively, that contained 10 % foetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin. Before the preparation of the Pb2+ cell lysis, the cells were cultured in the medium with different concentrations of Pb2+ for 24 h. Then, approximately 106 cells were resuspended in 1 mL of ice-cold sterile water, and cell lysis was performed through repeated freezing and thawing. When not in use, the cell lysate was kept in -80 °C refrigerator. Fabrication of the As-proposed Biosensor. The biosensor was fabricated by modifying nucleotides on the electrode surface layer by layer to form stable reticular DNA formation with abundant DNAzyme and intercalating [Ru(dcbpy)2dppz]2+ into the double-stranded DNA assembly. Typically, before modifying the GCE with nucleotides, it was first polished carefully with alumina slurries (0.3 and 0.05 µm) to remove the physically adsorbed materials, and then it was electrodeposited in HAuCl4 solution (10 mg/mL, -0.2 V, 30 s) to modify gold nanoparticles (AuNPs) on the GCE surface. Then, the AuNPs-modified GCE (AuNPs/GCE) was dipped in the 7



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capture linker solution at 4 °C for 12 h to absorb the capture linker onto the AuNPs surface based on the specific Au-S interaction between the SH terminal in the capture linker and AuNPs. Before the modification of S1 and S2 to form reticular DNA formation, S1 and S2 were first hybridized with assisted linker (AL) to form S1-AL and S2-AL, respectively. Then, the prepared S1-AL was modified onto the electrode surface via hybridization between the capture linker and S1-AL at 37 °C for 1 h. Based on the specific interaction between S1-AL and S2-AL, the S1-AL and S2-AL layers were modified onto the electrode surface layer by layer under similar conditions. Finally, the resulting electrode modified with reticular DNA formation was dipped in the solution with [Ru(dcbpy)2dppz]2+ at 4 °C for 12 h to intercalate [Ru(dcbpy)2dppz]2+ into double-stranded DNA assembly. After each modification, the resulting electrode was washed carefully with ultrapure water. Measurement Procedure. The ECL assay was performed based on the circular specific cleavage of the DNAzyme via Pb2+, and the ECL emission was measured with a multifunctional electrochemical and chemiluminescent analytical system in PBS with 25 mM TPrA. For measuring different samples, the proposed biosensor was dipped into the detection sample to cleave the reticular DNA formation through specific Pb2+-driven cleavage. With the Pb2+-driven cleavage, the reticular DNA formation would be disintegrated, and the ECL indicators, [Ru(dcbpy)2dppz]2+, would be dissociated from the electrode surface, which results in a decreased ECL emission signal associated with Pb2+ concentration. ■RESULTS AND DISCUSSION Characterization for the Fabrication of the Biosensor. In this study, the electrochemical measurements, including cyclic voltammetry (CV) and electrochemical impedance spectroscopy 8


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(EIS), were employed first to characterize the fabrications of the biosensor. The CV measurements were performed in 0.1 M PBS with 5 mM [Fe(CN)6]3-/4- at a potential range from 0.2 to 0.6 V (Figure 1A). As shown in the CV measurements, a pair of well-defined redox peaks of [Fe(CN)6]3-/4- were obtained for the bare GCE (curve a). After the electrodeposition of gold nanoparticles (AuNPs) onto an electrode surface, the oxidation-reduction currents were increased significantly with a decreased oxidation-reduction potential, which indicated there was good electron transfer on the electrode surface due to the good conductivity and electron transfer ability of AuNPs (curve b, AuNPs/GCE). When the capture linker was modified on the electrode surface by a specific interaction between the SH terminal in the capture linker and AuNPs (CL/AuNPs/GCE), the peak currents decreased because the nonconductive nucleotide sequences decreased electron transfer on the electrode surface (curve c). For the same reason, further decreases in peak currents were obtained after the hybridization of S1-AL and S2-AL layers, including S1-AL/CL/AuNPs/GCE (curve d), S2-AL/S1-AL/CL/AuNPs/GCE (curve e), S1AL/S2-AL/S1-AL/CL/AuNPs/GCE (curve f) and S2-AL/S1-AL/S2-AL/S1-L/CL/AuNPs/GCE (curve g). When the ECL indicators, [Ru(dcbpy)2dppz]2+ were intercalated into the doublestranded DNA of the reticular DNA formation (curve h, RU/S2-AL/S1-AL/S2-AL/S1AL/CL/AuNPs/GCE), the peak currents increased obviously, and the oxidation-reduction potential decreased significantly because the intercalated [Ru(dcbpy)2dppz]2+ would increase the conductivity of the reticular DNA formation on the electrode surface. Another important electrochemical measurement, EIS, was also employed to characterize the fabrications of the biosensor in 0.1 M PBS with 5 mM [Fe(CN)6]3-/4- at frequencies ranging from 5 × 10-2 to 1 × 106 Hz (Figure 1B), in which the electron transfer resistance (Ret), as a useful

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parameter to quantitatively demonstrate the electron transfer kinetics was investigated via the equicalent circuit with model impedance data. An EIS response with Ret of 52.3 Ω was obtained for the bare GCE (curve a). When the AuNPs were electrodeposited on the electrode surface (curve b, AuNPs/GCE), a significantly decreased Ret (Ret = 19.6 Ω) was observed due to the enhanced surface area and good conductivity of AuNPs on the electrode surface. After adding capture linker to AuNPs (curve c, CL/AuNPs/GCE), the Ret was increased to 41.8 Ω. When the S1-AL and S2-AL were hybridized onto the electrode surface layer by layer including S1AL/CL/AuNPs/GCE (curve d), S2-AL/S1-AL/CL/AuNPs/GCE (curve e), S1-AL/S2-AL/S1AL/CL/AuNPs/GCE (curve f) and S2-AL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE (curve g), the Ret values increased to 178.2 Ω, 285.6 Ω, 369.3 Ω and 422.9 Ω, respectively, due to the decreased electron transfer on the electrode surface via nonconductive nucleotide sequences. After the intercalation of [Ru(dcbpy)2dppz]2+ into the double-stranded DNA of the formed reticular DNA formation (curve h, RU/S2-AL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE), the electron transfer on the electrode surface increased obviously (Ret = 96.7 Ω). The trend of the electron transfer obtained from EIS results correlated with the CV results, which indicates successful fabrication of the biosensor.

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Figure 1 Characterizations for the fabrications of the proposed biosensor based on CV (A) and EIS (B); curve a, bare GCE; curve b, AuNPs/GCE; curve c, CL/AuNPs/GCE; curve d, S1AL/CL/AuNPs/GCE; curve e, S2-AL/S1-AL/CL/AuNPs/GCE; curve f, S1-AL/S2-AL/S1AL/CL/AuNPs/GCE; curve g, S2-AL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE and curve h, RU/S2-AL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE. These CV and EIS measurements provided important and useful characterization for the fabrication of the proposed biosensors, whereas just an indirect electron transfer tendency was observed via these measurements. To characterize surface morphology changes during fabrication of the biosensor, a technique using versatile methodology called AFM was used as well (Figure 2), which involved using an extremely high magnification microscope to analyze and characterize the surface at a microscopic level. As shown in Figure 2A for the surface morphology of the AuNPs-modified GCE, some mountain-like structure could be observed with increased surface area. After adding the capture linker to the AuNPs surface (Figure 2B), a smoother surface was obtained, which indicated the encapsulation of the capture linker on

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AuNPs via a specific Au-S interaction between the SH terminal in capture linker and AuNPs. After the construction of the reticular DNA formation on the electrode surface via hybridization of S1-AL and S2-AL layer by layer to form S1-AL/CL/AuNPs/GCE (Figure 2C), S2-AL/S1AL/CL/AuNPs/GCE (Figure 2D), S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE (Figure 2E) and S2AL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE (Figure 2F), the electrode surfaces gradually became smoother due to the formation of a DNA micronet on the AuNPs surface, which provided a good platform for an analytical assay. All of these CV, EIS and AFM results confirmed successful fabrication of the proposed biosensor.

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Figure 2 AFM images of the electrode surface with different modifications; A, AuNPs/GCE; B, CL/AuNPs/GCE; C, S1-AL/CL/AuNPs/GCE; D, S2-AL/S1-AL/CL/AuNPs/GCE; E, S1-AL/S2AL/S1-AL/CL/AuNPs/GCE and F, S2-SL/S1-AL/S2-AL/S1-AL/CL/AuNPs/GCE.

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ECL Performance and Mechanism of the Proposed ECL Strategy. The ECL performance (solid line) associated with electrochemical (dot line) response of the proposed biosensor is shown in Figure S3. An obvious ECL emission occurred on 1.3 V with the oxidation of [Ru(dcbpy)2dppz]2+ and TPrA, which indicates that the [Ru(dcbpy)2dppz]2+ has been intercalated into the double-stranded DNA of the reticular DNA formation and stably covered the electrode surface. Further characterization of the ECL emission via spectral analysis (Figure 3A-C) showed the maximum ECL emission on 609 nm, which was similar to the values reported for the ECL emission of [Ru(dcbpy)2dppz]2+ with TPrA as coreactant. Further measurements were obtained using fluorescence spectroscopy analysis (Figure 3D and E), the maximum excitation wavelength and emission wavelength for [Ru(dcbpy)2dppz]2+ were 548 nm and 609 nm, respectively. As shown in Figure 3F with the UV-vis adsorptions of [Ru(dcbpy)2dppz]2+, there was an obvious adsorption peak on approximately 450 nm. Based previous results and some related literature27-30, the basic ECL reactions include three main reactions (Scheme S1): (1) the oxidation of [Ru(dcbpy)2dppz]2+ to [Ru(dcbpy)2dppz]3+ and the coreactant (TPrA) to radical cation (TPrA•+); (2) the generation of [Ru(dcbpy)2dppz]*2+ by the reaction between [Ru(dcbpy)2dppz]3+ and TPrA•; and (3) ECL emission with [Ru(dcbpy)2dppz]*2+ backing to [Ru(dcbpy)2dppz]2+. These reactions could be simplified in the following equations: [Ru(dcbpy) dppz] − e → [Ru(dcbpy) dppz] TPrA − e → TPrA TPrA → TPrA + H [Ru(dcbpy) dppz] + TPrA → [Ru(dcbpy) dppz]∗

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[Ru(dcbpy) dppz]∗ → [Ru(dcbpy) dppz] + hγ

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Figure 3 (A) ECL-potential-wavelength results of the fabricated biosensor in PBS with 25 mM TPrA as coreactant via a 3D surface image model; (B) ECL-potential response of [Ru(dcbpy)2dppz]2+ with maximum emission wavelength on 609 nm; (C) maximum ECL spectroscopy of [Ru(dcbpy)2dppz]2+ with potential on 1.3 V; (D) normalized photoluminescence characterization of [Ru(dcbpy)2dppz]2+ based on 3D scan fluorescence spectroscopy via a 3D surface image model; (E) maximum fluorescence spectroscopy of [Ru(dcbpy)2dppz]2+ with a maximum emission wavelength on 609 nm and a maximum excitation wavelength on 548 nm, respectively; (F) UV-vis adsorption spectra characterization of the ECL indicator, [Ru(dcbpy)2dppz]2+ (1 mM in PBS). Optimization of Experimental Conditions. In target-driven circular cleaving, reaction time is the primary factor that influences the cleaving effect, ECL response and the assay performances, including the detection sensitivity and linear range. To investigate the optimal reaction time, the ECL responses of the proposed biosensor incubated with different concentrations of Pb2+ and reacted for different reaction time were recorded. As shown in Figure 4, the ECL intensity was decreased with the concentrations of Pb2+ ranging from 50 pM to 500 µM and the reaction times ranging from 30 s to 1830 s. Importantly, the detection sensitivity and linear range were influenced significantly by the reaction time. Considering the requirements for a normal concentration range, an optimized reaction time of 990 s was used for further experiments.

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Figure 4 (A) ECL responses of the proposed ECL assay with reaction time ranging from 30 s to 1830 s and concentration of Pb2+ rangeding from 50 pM to 500 µM; (B) ECL responses of the proposed ECL assay in a linear range with different reaction times. Application of the Proposed ECL Strategy. In this study, the performances of the proposed ECL assay for quantitative identification of Pb2+ were estimated by determining changes that occurred with different concentrations of Pb2+. As shown in Figure 5A, the ECL intensity decreased with the concentration of Pb2+ in a range from 50 pM to 500 µM. The linear relationship between the ECL intensity and concentration of Pb2+ could be demonstrated as

!"

# 12493 − 1364.5 - lg01234 with a detection limit of 4.73 pM, indicating the good

sensitivity of the proposed ECL assay, which should be due to the large immobilized amount of ECL indicators in the layered DNA micronet-carrier and target-driven circular etching for signal amplification. Additionally, the analytical performances of the proposed ECL strategy were compared with some previously reported assays, as shown in Table S1.

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Figure 5 (A) Calibration curve for the relationship between ECL intensity of the proposed ECL assay and concentration of Pb2+ with a reaction time of 990 s; (B) Specificity evaluation of the proposed ECL assay for 50 µM Pb2+, 100 µM interference metal ions, including Mg2+, Zn2+, Cd2+, Mn2+, Cu2+, Ni2+, Co2+, Fe3+, Ca2+ and Hg2+, and a mixture of Pb2+ and the interference metal ions; (C) Stability evaluation of the proposed ECL assay for 400 nM Pb2+. Evaluating the specificity of the proposed ECL strategy as an important property of an analytical method was performed by the detecting different metal ions, including Mg2+, Zn2+, Cd2+, Mn2+, Cu2+, Ni2+, Co2+, Fe3+, Ca2+ and Hg2+ with concentrations of 100 µM. As shown in Figure 5B, there were no obvious responses for these reference ions, and even a longer reaction time and higher concentrations were employed for these reference ions, which indicates the acceptable specificity of the proposed ECL strategy. Furthermore, the stability and repeatability of the proposed ECL strategy was evaluated in this study as well (Figure 5C and Figure S5), which was performed with the variation coefficients (CV%) for duplicate measurements. As expected, all of the CV% were not greater than 5%, which indicated the satisfactory repeatability and stability of the proposed ECL assay.

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Additionally, further tests of the applicability of the proposed ECL assay were performed based on the Pb2+ assay for the real samples, including measurements of Pb2+-polluted water and cells (Figure 6 and Figure S6). As shown in Figure 6A, the detection of Pb2+ in water based on the standard addition method had a correlation coefficient and slope that were close to 1, which indicates the proposed ECL biosensor was suitable for Pb2+ detection in water. Further application studies for the detection of Pb2+ in cells were performed based on the comparison between the proposed ECL assay and the conventional strategy, which is inductively coupled plasma mass spectrometry (Figure 6B). Although the concentration range was different from the concentration of Pb2+ in water, the lab study results for the proposed ECL assay had good agreement with the results of inductively coupled plasma mass spectrometry, indicating the good performance of the proposed ECL assay as an efficient approach for the determination of Pb2+.

Figure 6 Applicability evaluation of the proposed ECL assay based on the standard addition method (A) and comparison with the conventional strategy, inductively coupled plasma mass spectrometry (B).

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■CONCLUSION In summary, a target-driven circular cleavage-based ECL assay was proposed for ultrasensitive estimation of Pb2+ in water and cells by generating a multi-interface DNA micronet-carrier via hybridization of a double-stranded DNAzyme substrate layer-by-layer to immobilize amounts of ECL indicator, [Ru(dcbpy)2dppz]2+ in double-stranded DNA. When the double-stranded structures were cleaved circularly via Pb2+ in the detection sample, the ECL indicator could be released, which resulted in a decreased ECL signal associated with the concentration of Pb2+ having a higher sensitivity and a wider linear range. Further performances for the applicability of the proposed ECL assay demonstrated good agreement with additional Pb2+ and the conventional strategy, indicating the potential practical application value of the proposed strategy. Importantly, the success in establishing the proposed ECL assay offered a simple and efficient strategy for detecting various targets, including ions, proteins, nucleotide sequences and cells, which provides a new avenue for developing environmentally safe assay and clinical diagnostics. ■ASSOCIATED CONTENT ■SUPPORTING INFORMATION The Supporting Information is available free of charge on the ACS Publications website ad DOI: ***** Supplementary figures and tables for schematic illustration for the mechanism of the ECL reactions, CV-ECL performance, characterization of the oligonucleotides by polyacrylamide gel

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electrophoresis, characterization of ECL biosensor with different layers, investigation of the repeatability of the proposed ECL biosensor via intra- and inter-assay and comparison of the developed ECL strategy with other methods from the literatures. ■ABBREVIATION LIST AFM

atomic force microscopy

AL

assisted linker

AuNPs

gold nanoparticles

Cdl.

double-layer capacitance

CL

capture linker

CV

cyclic voltammetry

CV%

variation coefficients

DMEM

Dulbecco’s modified eagle’s medium

ECL

electrochemiluminescent

EIS

electrochemical impedance spectroscopy

FBS

fetal bovine serum

GCE

glassy carbon electrode

HAuCl4

gold chloride

ICP

inductively coupled plasma mass spectrometry

Pb

2+

lead (II) ions

Pb(NO3)2

lead nitrate

PBS

Phosphate buffered saline

Ret

electron transfer resistance

[Ru(dcbpy)2dppz]2+, RU

bis (4, 4'-dicarboxy-2, 2'-bipyridyl) (4, 5, 9, 14-tetraaza-benzo [β]triphenylene) ruthenium (II)

Rs

electrolyte resistance

TPrA

trimethylamine

Zw

Warburg impedance

■AUTHOR INFORMATION 21



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Corresponding Author * E-mail: [email protected] (Yaqin Chai), [email protected](Ruo Yuan). Notes The authors declare they have no competing financial interests. ■ACKNOWLEDGMENT This work was financially supported by the National Natural Science Foundation (NNSF) of China (21575116, 81301518, 51473136 and 21275119) and the Fundamental Research Funds for the Central Universities (XDJK2015A002), China. ■REFERENCES (1) Information for Workers: Health Problems Caused by Lead. The National Institute for Occupational Safety and Health, 2017. (2) Lead Poisoning and Health. World Health Organization, 2017. (3) Lead and Copper Rule State File Review: National Report. Environmental Protection Agency, U.S. Government Printing Office, Washington, DC, 2007. (4) Weyerman M, Brenner H. Factors Affecting Bone Demineralization and Blood Lead Levels of Postmenopausal Women. A population Based Study from Germany. Environ. Res. 1998, 76:19-25. (5) Zhang, X. B.; Wang, Z. D.; Xing, H.; Xiang, Y.; Lu, Y. Catalytic and Molecular Beacons for Amplified Detection of Metal Ions and Organic Molecules with High Sensitivity. Anal. Chem. 2010, 82, 5005-5011. (6) Wang, H. L.; Ou, L. M. L.; Suo, Y.; Yu, H. Z. Computer-readable DNAzyme Assay on Disc

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Route Involving TPrA•+ Cation Radicals. J. Am. Chem. Soc., 2002, 124, 14478-14485.

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A graphic entry for the Table of Contents (TOC)

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Electrochemiluminescent Pb2+-Driven Circular Etching Sensor

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