Surface Enhanced Electrochemiluminescence for Ultrasensitive Detection of Hg2+

Electrochimica Acta 150 (2014) 123–128

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Electrochimica Acta journal homepage: www.elsevier.com/locate/electacta

Surface Enhanced Electrochemiluminescence for Ultrasensitive Detection of Hg2+ Daifang Wang, Longhua Guo *, Rong Huang, Bin Qiu, Zhenyu Lin, Guonan Chen Institute of Nanomedicine and Nanobiosensing, Ministry of Education Key Laboratory of Analysis and Detection Technology for Food Safety,College of Chemistry, Fuzhou University, Fuzhou, 350116, China

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 July 2014 Received in revised form 23 September 2014 Accepted 24 October 2014 Available online 4 November 2014

Electrochemiluminescence (ECL) of Ru(bpy)32+ has been widely used in analytical chemistry. Herein, we present a new approach to enhance the ECL of Ru(bpy)32+ by the localized surface plasmon resonance (LSPR) of gold nanorods (AuNR). Our investigations reveal that the ECL intensity could be greatly enhanced by controlling the distance between Ru(bpy)32+ and the surface of AuNRs. We call this kind of surface plasmon induced ECL enhancement as surface-enhanced electrochemiluminescence (SEECL). This kind of SEECL phenomenon is utilized to fabricate a biosensor for ultrasensitive Hg2+ detection. The SEECL biosensor is fabricated by self-assembling AuNRs and T-rich ssDNA probes on gold electrode surface. With the presence of Hg2+, the conformation of ssDNA probes changed to be hairpin-like structure via formation of T-Hg2+-T structure. And Ru(bpy)32+ could insert into the grooves of the hairpin structured DNA probes to generate ECL emission, which could be enhanced by the LSPR of AuNR. The ECL intensity of the sensor increased with the concentration of Hg2+, and a detection limit of 10 fM Hg2+ in aqueous solution was achieved. The effect of different LSPR peak location of AuNR on the sensitivity of biosensor has been investigated. The results show that a good overlap between the LSPR absorption spectrum and the ECL emission spectrum of Ru(bpy)32+ could achieve the best ECL signal enhancement. ã 2014 Elsevier Ltd. All rights reserved.

Key words: Electrochemiluminescence LSPR Mercury Ru(bpy)32+ Surface Enhanced Spectroscopy

1. Introduction It is well-known that mercury is a virulent heavy metal for human health, and a global toxic pollutant for environment [1]. A majority of mercury comes from the effluent of industrial manufacture, and it exists in the water as the forms of organic mercury and inorganic mercury. Although, inorganic mercury,such as HgCl2, could be served as bactericide reagent for medicine [2], high concentration of solvable inorganic mercury in drinking water could corrode the stomach, intestines and kidney of human body, and it also could damage hearts of children [3], so that it is essential to detect solvable inorganic mercury in food and water. Up to present, the methods about mercury detection primary focusing on atomic spectroscopy (e.g. atomic adsorption spectroscopy (AAS) [4a,b,c], atomic emission spectroscopy (AES) [5], atomic fluorescence spectroscopy (AFS) [6], high performance liquid chromatography (HPLC) [7a,b], inductively coupled plasmon-mass spectroscopy (ICP-MS) [8a,b,c] and fluorescence analysis[9a,b,c].

* Corresponding author. Fax: +86 591 22866135. E-mail address: [email protected] (L. Guo). http://dx.doi.org/10.1016/j.electacta.2014.10.121 0013-4686/ ã 2014 Elsevier Ltd. All rights reserved.

ECL as a simple and sensitive analytical technology, has been applied extensively in the area of environmental, biochemical and pharmaceutical research [10a,b]. Up until now, a large quantity of ECL reagents have been developed. The most representative ECL reagents are Ruthenium(II) tris(bipyridine) (Ru(bpy)32+) and its derivatives due to their good water solubility, high electrochemical stability and the repeatedly regenerated ability in ECL reaction [11a,b]. Hitherto, researchers have explored a variety of ways to enhance the ECL emission of Ru(bpy)32+ for obtaining high sensitivity, for example, the development of new coreactant for the ECL of Ru(bpy)32+ [12], exploring the effect of different additives to the ECL emission of Ru(bpy)32+-TPrA system [13a,b], and the synthesis of dual-core or multi-core ruthenium complexes [14a,b]. However, up to now, few attention has been paid to the utility of LSPR of noble metal nanoparticles to enhance the ECL of Ru(bpy)32+. LSPR is an physical phenomenon occurred when the plasmon on the noble nanoparticle surface has the same oscillation frequency to the incident light. LSPR could generate a strongly enhanced localized electromagnetic field around the noble nanoparticles, which is the key for surface enhanced spectroscopy such as surface enhanced Raman spectroscopy and surface enhanced fluorescence [15a,b]. Recently, researchers revealed that the ECL

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intensity of CdS Nancrystals could be significantly improved by LSPR of noble metal nanoparticles [16a,b,c]. Herein, we would like to explore the possibility of use LSPR of AuNRs to enhance the ECL of Ru(bpy)32+ since Ru(bpy)32+ is the most extensively used ECL reagent. In this paper, we developed a label-free SEECL biosensor for ultrasensitive detection of Hg2+. AuNRs were immobilized on the surface of the working electrode and then T-rich ssDNA probes were modified on the surface of AuNRs. In the presence of the target molecule (e.g. Hg2+), the T-rich ssDNA probes would form into a hairpin structure so that Ru(bpy)32+ was able to insert into the grooves of the hairpin DNA. The ECL signal of Ru(bpy)32+ intercalating in the hairpin DNA can be dramatically enhanced by LSPR of AuNR. However, in the absence of Hg2+, no hairpin DNA was formed, thus no ECL signal can be detected. Something unique in this work includes: i) The LSPR of AuNRs was used to enhance the ECL of Ru(bpy)32+ for the first time; ii) Our investigation revealed that the amount of ECL enhancement is closely related to the LSPR peak wavelength of AuNRs; and iii) The LOD for Hg2+ is 10 fM, the lowest one we have ever seen by an ECL sensor. 2. Materials and methods 2.1. Apparatus and Reagents The ECL measurements were recorded by a model BPCL-1-TIC ultra weak luminescence analyzer at room temperature, and the voltage of the PMT was set at 1000 V. The electrochemical measurements were detected with a CHI 660D electrochemical workstation (Shanghai CHI Instruments Co., China). All experiments were carried out with a conventional three-electrode system. The reference electrode was a Ag/AgCl electrode, working electrode was a AuNR-modified gold electrode (GE, w = 2 mm), and a Pt wire served as the counter electrode. The UV–vis absorption spectra were obtained on a Shimadzu UV-3600 UV-vis-NIR photo spectrometer (Perkin-Elmer Co., USA). All DNA oligonucleotides (as shown in Table 1) were acquired from Sangon Biotech (Shanghai) Co., Ltd. Tris(2,20 -bipyridyl) dichlororuthenium (II) hexahydrate (Ru(bpy)3Cl26H2O) was purchased from Sigma-Aldrich. Hydrogen tetrachloroaurate tetrahydrate (HAuCl44H2O) and cetyltrimethyl ammonium bromide (CTAB) was purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Tri-n-propylamine (TPrA), N-hydroxysuccinimide (NHS) and L-ascorbic acid (VC) were purchased from Aladdin (Shanghai, China), and other reactants were obtained from Fuchen Chemical Reagents Ltd Company (Tianjin, China). All reagents were of analytical grade and used as received. Millipore ultrapure water (resistivity > 18.2 MV cm) was used throughout the experiment. 2.2. Preparation of AuNR AuNRs were synthesized by a seed-mediated method. Briefly, 0.6 mL 0.01 M fresh ice NaBH4 was injected to an aqueous solution containing 9.75 mL of 0.1 M CTAB and 0.25 mL of 0.01 M HAuCl4 under vigorous stirring to obtain the gold seed solution. Next, 40 mL 0.1 M CTAB, 2 mL 0.01 M HAuCl4 and 0.4 mL 0.01 M AgNO3

Table 1 Sequences of DNA probe used in this work. DNA name

Sequence

DNA1 DNA2 DNA3

5’-SH-C6-CCCTTTTTTTTTCCCCCCTTTTTTTTT-3’ 5’-SH-C6-CCCCCCCCCTTTTTTTTTCCCCCCTTTTTTTTT-3’ 5’-SH-C6-CCCCCCCCCCCCCCCTTTTTTTTTCCCCCCTTTTTTTTT-3’

The underline bases are utilized as the spacers.

were add into a 50 mL flask in turn. And 0.32 mL 0.1 M freshly prepared VC was injected into the mixture solution, followed by the addition of 0.8 mL 1.0 M HCl. At last, 0.01 mL gold seed solution was added. The mixture solution was left undisturbed for at least 6 h at room temperature. In order to obtain AuNRs with different aspect ratio, the asgrown AuNR sample was undergone an anisotropic oxidation process as follows: 0.8 mL 1.0 M HCl was injected into the as-grown AuNR solution, and then O2 was bubbled into the solution for 10 min. The mixture solution was incubated in a water bath at 65  C for a time duration varied from 2 hours to 24 hours to obtain AuNRs with different aspect ratio. Finally, the AuNR solution was centrifuged at 8500 rpm for 10 min and the precipitate was redispersed in H2O for further characterization. 2.3. Fabrication of SEECL Sensors Gold electrode was pretreated by polishing with 0.5 mm and 0.05 mm of alumina aqueous slurry successively, and washed with 50% (v/v) HNO3, 50% (v/v) ethanol and doubly distilled water in turn. Then the bare electrode was electrochemically cleaned by scanning in a potential ranging from -0.4 V to +1.6 V in 0.5 M H2SO4 solution for 20 times, and rinsed with doubly distilled water. Next, the gold electrode was immersed in 0.5 mM 3-mercaptopropionic acid (MPA) for 1 hour, and 0.1 mM b-mercaptoethanol (b-ME) for 30 min, respectively. This procedure would generate a dense MPA monolayer at the surface of the gold electrode (GE/MPA). The carboxyl was then activated by immersing the electrode into a mixture solution of 0.4 mM EDAC and 0.1 mM NHS for 1 hour. After careful washing with water, the electrode was dipped in AuNR aqueous solution to obtain the AuNR modified gold electrode (GE/ MPA/AuNR). Next, 2 mL 0.1 M PH 7.4 Tris-HCl-NaCl solution containing 0.1 mM ssDNA probe was dropped on the surface of the modified electrode, and incubated at room temperature for 30 min to obtain the DNA probe modified gold electrode (GE/MPA/ AuNR/DNA). The modified electrode was washed carefully with 0.1 M PH 7.4 Tris-HCl-NaCl solution to remove the unbound ssDNA probes, and stored at 4  C. 2.4. ECL Measurements The sensor was immersed in 0.1 M pH 7.4 Tris-HCl-NaCl solution containing different concentration of HgCl2 for 30 minutes at 37  C, followed by dropping 2 mL 1.0 mM Ru(bpy)32+ on the surface of modified electrodes to allow the binding of Hg2+ to the probe DNAs. This Hg2+ modified electrode is called GE/MPA/AuNR/DNA/Hg2+ hereafter. Then the biosensors was dried at 4  C, and electrochemical cleaned in 0.1 M PH 7.4 PBS by scanning from -0.5 V to +1.5 V via cyclic voltammetry. ECL measurements of biosensors were obtained in 0.1 M PH 7.4 PBS containing 1 mM TPrA with a scanning rate of 0.1 V/s. 3. Results and Discussion 3.1. Principle of the SEECL biosensor A schematic diagram of the proposed SEECL biosensor is shown in scheme 1. AuNRs were firstly immobilized on the surface of the gold electrode via electrostatic assembly. Then the single stranded T-rich DNA probes was bound to AuNR surface by Au-S interaction. In the presence of Hg2+, the T-rich ssDNA probe would form a multiple of T-Hg-T structures, which lead to conformation change of the ssDNA probes to a stem-loop structure. Ru(bpy)32+ could then insert into the grooves of the double stranded DNA (the stem of the hairpin DNA), and ECL could be generated. The ECL emission would generate surface plasmon at the surface of AuNRs as well.

D. Wang et al. / Electrochimica Acta 150 (2014) 123–128

Scheme 1. schematic diagram of SEECL biosensor for Hg

This kind of strong electromagnetic oscillation could effectively improve the ECL efficiency of Ru(bpy)32+, hence the ECL intensity could be greatly enhanced.

2+

125

detection.

+1.2 V on CV curve in Fig. S1 B belong to the redox reaction of Ru (bpy)32+, which indicated the successful insertion of Ru(bpy)32+. 3.3. LSPR enhanced ECL

3.2. Electrodes characterization The modification processes of different electrodes were observed via electrochemical impedance spectroscopy and cyclic voltammetry (CV) (See Figure S1). Figure S1 A depicts the distinct electrochemical impedance spectroscopy (EIS) of different modified electrodes. It could be observed from the Nyquist plots that the electron transfer resistance (Rct) at the electrode surface changed after modification. The increase of Rct is because that the electron transfer of MPA and DNA is worse than gold. Besides, after the binding of Hg2+ to the working electrodes, significant Rct increase is observed, which is attributed to the formation of duplex DNA by THg-T structure. On the other hand, current peak located at about

In order to show the ECL enhancement of AuNRs, we compared the ECL emission of GE/DNA/Ru(bpy)32+ and GE/AuNR/DNA/Ru (bpy)32+ in the same PBS solution containing 1.0 mM TPrA (Fig. 1A). The results showed that the ECL intensity of electrode modified with AuNR was much higher than the one without AuNR. It is worth to note that the simultaneous electrochemical measurements of the two electrodes was nearly identical (See Fig. 1B). So that ECL signal amplification by electrochemical effect via increase of electrode surface area after AuNR modification, which was named as nano-electrode effect, should be ignored. Thus this kind of greatly ECL enhancement should be mostly attributed to the LSPR of AuNRs rather than the electrochemical effect.

Fig. 1. ECL intensity (A) and the simultaneous electrochemical measurements (B) of two different electrodes: GE/DNA/Ru(bpy)32+(black) and GE/AuNR/DNA/Ru(bpy)32+ (red) in the same 0.1 M PH 7.4 PBS containing with 1.0 mM TPrA; the scanning rate is 100 mV/s.

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Fig. 2. (A) TEM images of AuNR samples; (B) UV–vis spectra of different as synthesis AuNR: AuNR-1 (green), AuNR-2 (red) and AuNR-3 (black); (C) Relationship between ECL intensity and the longitudinal LSPR band of AuNRs.

3.4. Influence of LSPR peak location to the ECL enhancement LSPR peak location of AuNRs is closely related to their aspect ratio. Herein we firstly synthesized AuNRs with relatively large aspect ratio, and then shorted the AuNRs by an anisotropic oxidation approach. Typical TEM images of these AuNR with different aspect ratio are shown in Fig. 2A. From the TEM images we could see that the aspect ratios of AuNR-1, AuNR-2 and AuNR3 were about 2:1 3:1 and 4:1, respectively. The LSPR spectra of

these AuNRs are shown in Fig. 3B. Each spectrum has two LSPR bands: one located at 520 nm, which belongs to the transverse LSPR band of rod-like nanoparticles; the other peak belongs to the longitudinal LSPR band of rod-like nanoparticles, which is changed with the corresponding aspect ratio. As shown in Fig. 2B, the longitudinal LSPR peak location for AuNR-1, AuNR-2 and AuNR3 are at 607 nm, 632 nm and 673 nm, respectively. Next, we investigated the influence of AuNR longitudinal LSPR band on the ECL intensity, and the results were shown in Fig. 2C. In

A

B

6000

40

2+

20

DNA1 DNA2 DNA3

A

4000

Current /

ECL Intensity / a.u.

0 M Hg 2+ 1.0 M Hg

2000

0

-20

0 DNA1

DNA2

DNA3

Types of DNA probes

-0.5

0.0

0.5

1.0

1.5

Potential / V (vs Ag/AgCl)

Fig. 3. ECL intensity (A) and the simultaneous electrochemical measurements (B) of electrodes modified with different ssDNA probe: DNA 1 (black), DNA 2 (red) and DNA 3 (green). ECL measurements were conducted in a solution containing 0.1 M PH7.4 PBS and 1.0 mM TPrA; the scanning rate is 100 mV/s.

D. Wang et al. / Electrochimica Acta 150 (2014) 123–128

B

A 6000

3000

ECL Intensity / a.u.

ECL Intensity / a.u.

127

2+ Hg concentration

4000

-6 1X10 M

2000

2000

1000

0M

0 0.0

0.5

1.0

1.5

Potential / V (vs Ag/AgCl)

0 -14

-12

-10

-8

Loc C Hg 2+ / (mol/L)

Fig. 4. (A) Concentration dependent ECL behaviors of the sensor. Hg2+ from top to toe is: 1.0  106 M, 1.0  107 M, 1.0  108 M, 1.0  109 M, 1.0  1010 M, 1.0  1011 M, 1.0  1012 M, 1.0  1013 M and 1.0  1014 M, respectively; (B) The calibration curve for quantification of Hg2+. The ECL buffer containing 0.1 M pH 7.4 PBS and 1.0 mM TPrA. The scanning rate is 100 mV/s for all measurements.

order to exclude the effect of electrochemical enhancement, we intentionally controlled the surface modification of each electrode so that the electrochemical response of each electrode is identical (See Fig. S2). It is obvious that the ECL intensity of electrode modified with AuNR-2 has the highest value compared with the other two. The reason may be due to that the ECL emission wavelength of Ru(bpy)32+ is about 630 nm, which was close to the longitudinal LSPR peak wavelength of AuNR-2. Therefore, we inferred that best ECL enhancement could be obtained in condition that there is a good overlapping between the ECL emission of luminophore and the LSPR band of noble metal nanoparticles. 3.5. Influence of spacer length of the DNA probes There are two factors that affect the ECL emission of Ru(bpy)32+: i) energy transfer between the excited-state of Ru(bpy)32+ and AuNRs, which would generate ECL quenching; and ii) LSPR induced ECL enhancement. It is worth to note that both the energy transfer generated ECL quenching and the LSPR induced ECL enhancement are sensitive to the inter-distances between Ru(bpy)32+ and AuNRs. Thus it is vital important to control the inter-distances between Ru (bpy)32+ and AuNRs to achieve best ECL enhancement. We purchased three different DNA oligonucleotides as sensor probes, and their sequences were listed in Table 1. The only difference among these ssDNA probes is the quantity of C base located at the 50 end, which is used as the spacers. The number of C base decided the reaction distance between Ru(bpy)32+ and gold nanoparticles. Thus, the distance relationship among ssDNA probe is DNA1 < DNA2 < DNA3. Maximum ECL intensity was observed when the electrode was modified with DNA2 probe (Fig. 3A). This kind of differences in ECL response should mainly come from the difference in the inter-distance between Ru(bpy)32+ and gold nanoparticles. The reaction distance for the electrode modified with DNA1 has the shortest inter-distance. in this case, part of Ru (bpy)32+ imbedded in the DNA1 probe were so close to the surface of AuNR that energy transfer occurred. This kind of energy transfer would generate the quenching of ECL, thus the observed ECL intensity is the lowest; While the reason that the ECL intensity of electrode modified with DNA 3 was less than those modified with DNA 2 because that the inter-distance between Ru(bpy)32+ and

gold nanoparticles is too far away from the surface of AuNR. It is well-known that the electromagnetic fields of noble metal nanoparticles are decaying exponentially with the distance. As a result, the ECL enhancement effect weakened. 3.6. Selectivity of the sensor In order to show the specific of the sensor for Hg2+, we investigated the responses of this sensor to other metal ions such as Pb2+, Cu2+, et al. (Fig. S3). Fig S3 is ECL intensity of different metal ions modified electrode in the same 0.1 M PH 7.4 PBS containing 1.0 mM TPrA. The results showed that the ECL intensity was more or less enhanced after the binding of different metal ions. However, it is clearly seen that the ECL response of Hg2+ (1.0 mM) is much higher than other types of metal ions even at a concentration of 100 times of that of Hg2+ (100 mM). The results indicated that this type of biosensor has good selectivity for the determination of Hg2 + . 3.7. Hg2+ Detection We investigated the concentration dependent response of the sensor by testing a series of standard solution containing different Hg2+ concentration varied from 10.0 fM to 1.0 mM, and the results are shown in Fig. 4A. The lowest detectable concentration of Hg2+ is 10.0 fM. Linear response is observed from 10 fM to 10.0 pM (See Fig. 4B). The linear relation coefficient is 0.9964, and the linear relation equation is 4I = 6364 + 435.7  logC (Hg2+). We repeated the same experiment in contaminative water by SEECL biosensor for 6 times, and the results was shown in figure S4. The average detection concentration of Hg2+ in contaminative water is 2.75 pM, and the RSD is 6.14%. While the detection Hg2+ concentration of the same one contaminative water by ICP-MS is 2.79 pM, which is in good consistent with the results obtained from the proposed sensor. 4. Conclusion In summary, this work demonstrates a SEECL DNA biosensor for ultrasensitive detection of Hg2+. In the presence of Hg2+, the DNA

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probe formed into a hairpin loop by the formation of T-Hg-T structure. Ru(bpy)32+ inserted into the grooves of this hairpin DNA to produce an ECL signal. AuNRs were used to enhance the ECL intensity. Our investigation revealed that maximum ECL enhancement was achieved when the LSPR band had a good overlapping with the ECL emission spectrum of Ru(bpy)32+. Under the optimal condition, the proposed ECL sensor could selectively detect Hg2+ and the LOD is 10 fM. This is the lowest LOD for Hg2+ we have ever seen by an ECL based sensor. This sensor could have potential application for the detection of trace amount Hg2+ in environmental samples. Acknowledgements The authors gratefully acknowledge the financial support of the 973 Program of China (2010CB732403), NSFC (21277025, 21205017), Natural Science Foundation of Fujian Province (2012J01036), the Foundation of Fujian Educational Committee (JA12039, JA13024), the Major project of Fujian Province (2011N5008), and the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.electacta.2014.10.121. Reference [1] K. Leopold, M. Foulkes, P. Worsfold, Methods for the determination and speciation of mercury in natural waters–a review, Analytica Chimica Acta 663 (2) (2010) 127–138. [2] T.W. Clarkson, L. Magos, G.J. Myers, The toxicology of mercury—current exposures and clinical manifestations, New England Journal of Medicine 349 (18) (2003) 1731–1737. [3] J. Risher, R. DeWoskin, Toxicological profile for mercury, Agency for Toxic Substances & Disease Registry (1999) . [4] (a) W.R. Hatch, W.L. Ott, Determination of submicrogram quantities of mercury by atomic absorption spectrophotometry, Analytical Chemistry 40 (14) (1968) 2085–2087; (b) L.B. Escudero, R.A. Olsina, R.G. Wuilloud, Polymer-supported ionic liquid solid phase extraction for trace inorganic and organic mercury determination in water samples by flow injection-cold vapor atomic absorption spectrometry, Talanta 116 (2013) 133–140; (c) V.A. Lemos, L.O. dos Santos, A new method for preconcentration and determination of mercury in fish, shellfish and saliva by cold vapour atomic absorption spectrometry, Food chemistry 149 (2014) 203–207. [5] X. Yuan, G. Yang, Y. Ding, X. Li, X. Zhan, Z. Zhao, Y. Duan, An effective analytical system based on a pulsed direct current microplasma source for ultra-trace mercury analysis using gold amalgamation cold vapor atomic emission spectrometry, Spectrochimica Acta Part B: Atomic Spectroscopy (2014) . [6] A.T. Reis, C.B. Lopes, C.M. Davidson, A.C. Duarte, E. Pereira, Extraction of mercury water-soluble fraction from soils: An optimization study, Geoderma 213 (2014) 255–260. [7] (a) C. Ibáñez-Palomino, J.F. López-Sánchez, À. Sahuquillo, Inorganic mercury and methylmercury determination in polluted waters by HPLC coupled to cold vapour atomic fluorescence spectroscopy, International Journal of Environmental Analytical Chemistry 92 (7) (2012) 909–921; (b) M. Wang, W. Feng, J. Shi, F. Zhang, B. Wang, M. Zhu, B. Li, Y. Zhao, Z. Chai,

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