Plasmon-Enhanced Electrochemiluminescence for Nucleic Acid

Dec 18, 2017 - DNA tetrahedron has a stable and rigid three-dimensional scaffold which can be changed through the mechanical reconfiguration of DNA st...
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Plasmon-Enhanced Electrochemiluminescence for Nucleic Acid Detection Based on Gold Nanodendrites Mei-Xing Li, Qiu-Mei Feng, Zhen Zhou, Wei Zhao, Jing-Juan Xu, and Hong-Yuan Chen Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b04307 • Publication Date (Web): 18 Dec 2017 Downloaded from http://pubs.acs.org on December 18, 2017

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Analytical Chemistry

Plasmon-Enhanced Electrochemiluminescence for Nucleic Acid Detection Based on Gold Nanodendrites Mei-Xing Li†, Qiu-Mei Feng†, Zhen Zhou, Wei Zhao*, Jing-Juan Xu*, and Hong-Yuan Chen State Key Laboratory of Analytical Chemistry for Life Science and Collaborative Innovation Center of Chemistry for Life Sciences, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210023, China. * Corresponding author. Tel/Fax: +86-25-89687294; E-mail address: [email protected] (W. Zhao) [email protected] (J.J. Xu) † These authors contributed equally to this work.

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ABSTRACT: Gold nanodendrites (Au NDs) exhibit extremely strong electromagnetic field located around multiple tip branches due to plasmon coupling effect. In this work, a novel LSPR enhanced ECL emission from CdTe nanocrystals (NCs) by Au NDs for the detection of nucleic acid is reported. This system is composed of a thin film of CdTe NCs on glassy carbon electrode (GCE) as anodic ECL emitter and Au NDs as plasmon enhancer. DNA tetrahedron embedded with a stem-loop hairpin structure on one edge was applied as a switch to regulate the distance between CdTe NCs and Au NDs. At original state, the hairpin structure was closed and DNA tetrahedron played in a relaxed state on CdTe NCs film. The ECL emission of CdTe NCs was quenched by proximal Au NDs due to Förster resonance energy transfer (FRET), which was defined as “turn-off” mode. After the complementary hybridization with target DNA, the hairpin structure changed to a rod-like configuration, resulting in an increased distance between CdTe NCs and Au NDs, and a significant enhancement of ECL induced by LSPR of Au NDs, which was defined as “turn-on” mode. Along with asymmetric modification method, a controllable and versatile pathway for modifying nanomaterials, the ECL sensor performed well with great stability and repeatability for nucleic acid detection in the range of 1.0 fM to 500 fM. Considering the high sensitivity and selectivity in the serum sample assay, this proposed method indicates a great potential for bioassay application.

In recent years, due to their prominent optical, electric and catalytic properties, noble metal (eg. Au, Ag) nanoparticles (NPs) have attracted considerable attention in fundamental researches and technical applications.1-4 One interesting topic relating to this area was the exploitation of non-spherical noble metal NPs since their optical behaviors were both size and shape dependent.5,6 For example, some non-spherical Au NPs with special superstructures (flower, star, dendrite, satellite, etc.) possessed extremely strong electromagnetic field according to the plasmon coupling between multiple branches. This property could bring many advantages for non-spherical Au NPs over spherical ones in analytical applications, such as colorimetric shift,7,8 localized surface plasmon resonance (LSPR) enhanced fluorescence,9 surface-enhanced Raman scattering (SERS)10-12 and so on. In 2009, our group firstly reported the distance-dependent quenching and enhancing of reductive-oxidative (R-O) ECL from semiconductor nanocrystals (NCs) based on LSPR of Au NPs.13 The distance-dependent enhancement of ECL signal was mainly caused by the energy transfer from ECL excited LSPR in Au NPs to NCs, and has become a good strategy for the development of highly sensitive biosensors. Later, our group further studied that Au NP dimers served as nanoantennas to mediate the distance-dependent LSPR enhanced ECL of CdS NCs.14 Due to the strong electromagnetic field induced by plasmon coupling in the nanogap between two NPs, an excellent enhancement of the ECL intensity was induced by Au NP dimers compared with that by monomer. This plasmon coupling strategy displayed potential application in ultra-sensitive bioassays. These studies reported above focused on the LSPR enhanced R-O ECL behavior of CdS NCs. For oxidative-reductive (O-R) ECL emitter, ECL from

Ru(bpy)32+ enhanced by Au NPs LSPR was verified by Wang and co-workers.15-17 Here, this plasmon-enhanced ECL phenomenon is further proved in another O-R ECL emitter, CdTe NC- gold nanodendrite (Au ND) system. With the co-reactant of tripropylamine (TPrA), CdTe NCs performed strong and stable ECL signals. While the distance between CdTe NCs and Au NDs were controlled properly, Au NDs bring in better performance for the influence of LSPR on ECL emission of NCs in the view of the significantly large local electromagnetic field enhancement that located around the sharp tips of nonspherical noble metal NPs. Due to its simple and precise self-recognition rule of G-C and A-T pairings, DNA was easy to hold multiple doublehelical domains together via the interchange of DNA backbones.18 Since the first report on the study of DNA nanotechnology by Nadrian Seeman in the 1980s,19 the exploiting and application of DNA nanostructure have become a rapidly growing and prosperous area. For example, based on the unique properties of three-dimensional scaffold, structural stability, well-defined spacing and specific orientation, DNA tetrahedral nanostructures have been splendid in the construction and application of electrochemical switching,20 intracellular logic sensors,21 biomolecular probe carrier platform,22 charge transferring framework,23 multiplex microarray platform24 and so on. In addition, DNA tetrahedron grafting on the electrode surface could effectively reduce the local overcrowding effect with well-defined spacing and bring in higher electrochemical and fluorescent sensitivities than linear DNA in biological detections.22,25,26 Recently, self-assembled tetrahedron DNA dendrimers were applied as nanocarriers to achieve a high loading efficiency for ECL luminophore with significantly amplified ECL signal output,27 and provided probe

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Scheme 1. (A) Stepwise asymmetric modification for Au NDs. (B) Schematic illustration of LSPR enhanced ECL sensor based on the DNA tetrahedral nanoswitch. DNA a solution-like phase for DNA assembly amplification,28 which bringing in a low detection limit and high sensitivity. Herein, an LSPR-enhanced O-R ECL emission sensor was designed using DNA tetrahedral scaffold as a switch to regulate the distance between CdTe NCs and Au NDs, while Au NDs acted as both ECL quencher and enhancer. DNA tetrahedron has a stable and rigid three-dimensional scaffold which can be changed through the mechanical reconfiguration of DNA strand at the edge of the tetrahedron in response to the external stimuli.17 As shown in Scheme 1, three-dimensional DNA tetrahedron with three vertices modified with amino groups was grafted on the CdTe NCs-modified electrode surface via amide bond. A stem-loop hairpin structure was inserted into one edge of DNA tetrahedron. When the hairpin was formed in its “closed” state, this edge was at the shortest length of 6 nm. In the present of target DNA, the hairpin structure stretched to rod-like by hybridizing both the stem and the loop, resulting in the DNA tetrahedral reconfiguration switched from relaxed state to taut state and reached to a large distance (12 nm) between CdTe NCs and carboxyl group at the vertex of DNA tetrahedron. Once carboxyl-terminated taut DNA tetrahedron was directly assembled with amineterminated Au NDs by carboxyl-amine attraction, the ECL intensity was in positive relationship with the distance between CdTe NCs and Au NDs. Finally, the concentration of target DNA was sensitively determined via the target-induced configuration changes of DNA tetrahedron based on the distance mediated LSPR-ECL. The phenomenon of LSPR induced ECL enhancement of CdTe NCs was similar to CdS NCs and Ru(bpy)32+. In addition, the experimental results and theoretical calculations both have verified that gold nanoparticles with different shapes produce strong electromagnetic field enhancements with different degrees which contribute efficiently to the signal enhancement in ECL sensing. Assisted with the asymmetric modification of gold nanoparticles, the

ECL signals obtained after modification on the surface of electrode performed well with great stability and repeatability. This controllable asymmetric functionalization provides a more versatile pathway for modifying nanomaterials, bring in great potential in the construction of biosensors for life analysis.

EXPERIMENTAL SECTION Reagents and Apparatus. Refer to the Supporting Information. Synthesis of NAC-CdTe NCs. The NAC-CdTe NCs were synthesized according to the literature with some modifications.29 Briefly, sodium hydrogen telluride (NaHTe) solution was prepared by the reaction of sodium borohydride (NaBH4) and tellurium with the molar ratio of 2:1 in ultrapure water under N2 atmosphere and magnetic stirring. NAC (15.0 mM) and CdCl2 (12.5 mM) were dissolved in 40.0 mL of ultrapure water under an ice-water bath, and the pH was carefully adjusted to 10.5 with NaOH solution (1.0 M). Then a fresh NaHTe solution at 0 °C, was added to the NAC and CdCl2 solution under vigorous stirring. The molar ratio of Cd:Te:NAC was fixed at 1.0:0.2:1.2. After reaction at room temperature for 10 min, the solution was transferred into a 40.0 mL Teflon-lined stainless steel autoclave and incubated in an oven at 200 °C for 70.0 min to afford the crude NACCdTe NCs. To remove the residual regents such as NAC, Cd2+, and Te2-, the crude NAC-CdTe NCs were then carefully purified through a triple preparation process with cold 2propanol. Finally, the as-prepared NAC-CdTe NCs were stored in ultrapure water for further experiment. Synthesis and Amine Asymmetric Modification of Au NDs. The synthesis of Au NDs was according to the previously reported methods with some modifications.9 Briefly, 200 µL of 5 % sodium citrate solution was added into 20 mL of 0.25 mM boiling HAuCl4 solution under stirring. The mixed solu-

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tion was allowed to run until the color became wine red, getting Au seeds. Then, to grow Au NDs, 2 mL of 0.25 mM HAuCl4 solution was adjusted to pH 11.5 by 1.0 M NaOH solution. Under slight stirring, the mixture of 16 µL NH2OH·HCl solution (40 mM) and 160 µL Au seeds was added into the above solution at the room temperature. The color of the solution changed from wine red to blue after 1 min, indicating the successful synthesis of Au NDs. Because the chlorine ions could further accelerate the intraparticle ripening of the Au nanodendrites,1 the final reaction solution was centrifuged to collect precipitates, and washed thoroughly with ultrapure water for three times. After that, the obtaining Au NDs precipitates were ultrasonically dispersed into ultrapure water for further use.

Figure 1. TEM images of (A) MCH and cystamine asymmetrically modified Au NDs, (B) sharp tip of the modified Au NDs, (C) Au NPs with the average diameter of 60 nm, and (D) NAC-CdTe NCs. (E) The UV-vis absorbance and fluorescence spectra of NAC-CdTe NCs. (F) The normalized intensity of (a) ECL spectra of CdTe NCs which were measured with optical filters (20 nm spaced), and the normalized UV-vis absorption of AuNPs (b) and AuNDs (c).

To achieve the Au NDs that modified with amine group on the top point, asymmetric modification was prepared according to previous report with some modification.30 The stepwise asymmetric modifying process for Au NDs was shown in Scheme 1. First, glass slides (25 mm × 9 mm) were cleaned in 30 % H2O2 solution (mixed in 1:3 ratio with concentrated H2SO4) for 30 min, followed by being washed thoroughly with distilled water and dried with nitrogen. Then, these clean glass slides were immersed in a (3-aminopropyl) triethoxysilane (APTES) solution (1 % v/v, prepared by ethanol) for aminosilanization overnight at 4 °C. After being washed with ethanol and ultrapure water, these APTES-modified glass slides were dried in an oven for 30 min at 120 °C. The silanized glass slides were immersed in a negative charge-capped Au NDs (Figure S1 Zeta potential of Au NDs and Au NPs) solution for

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6 h at room temperature based on electrostatic attraction, and then they were rinsed with water and placed in 10 µM 6mercapto-1-hexanol (MCH) solution overnight. After being washed with water, MCH-modified Au NDs were removed from glass slides via ultrasonic treatment in 1.0 mL water. 10 µM cysteamine was dropped into the above solution overnight to make the top point of Au NDs carrying with amine group. Finally, the asymmetrically modified Au NDs solution was centrifuged (7000 rpm, 6 min) and washed three times with ultrapure water, then resuspended in 1.0 mL PBS (10 mM KH2PO4-K2HPO4; pH 7.4) for the next use. Preparation of Asymmetrically Modified Au NPs. Au NPs with the average diameter of 60 nm were prepared as follows: after 50 mL of 0.01 % HAuCl4 solution was heated to 100 °C, 0.4 mL of trisodium citrate (1 %) was added to the above boiling solution under stirring. The reaction mixture was stirred for 20 min at 100 °C until the color turned purple red and then stored at 4 °C. The procedure of asymmetrically modified Au NPs was the same as that of Au NDs. Formation of DNA Tetrahedron. DNA tetrahedron was formed by one-pot incubation technique with a simple annealing process31,32 and the preparation process was shown in Scheme 1. First, four DNA strands (S1, S2, S3, S4) were diluted with 20 mM PBS buffer (pH 7.4) containing 5.0 mM Mg2+ to a common final concentration of 5 µM. Then, the obtained four DNA strands (S1, S2, S3 and S4, the sequences in the Table S1) were mixed in equimolar amounts in 20 mM PBS buffer (pH 7.4) containing 5.0 mM Mg2+, heated to 95 °C for 5 min and then cooled to room temperature overnight. Once assembled, the DNA tetrahedron contained one carboxyl group at one top and three amino groups at other three vertices. Finally, the obtained DNA tetrahedron solution was purified by ultrafiltration to remove the non-conjugated oligonucleotides. In addition, other DNA tetrahedral nanostructures (S2S3S4S5, S6S7S8S9) were assembled using the same procedure. Agarose Gel Electrophoresis. Different DNA structures (target DNA, S1, S2, S3, S4, S1S2S3, S1S2S4, S1S3S4, S2S3S4, S1S2S3S4) were all incubated at 95 °C for 5 min and then slowly cooled to room temperature overnight. The reconfigurable taut DNA tetrahedral scaffolds (S1S2S3S4-target DNA) were obtained through the incubation of target DNA and S1S2S3S4 for 90 min at room temperature. The 3 % agarose gels containing 3 µL of GelRed were prepared using 1× TBE buffer. Then, the loading samples with different DNA structures were injected into agarose gels and electrophoresis analysis was run at 110 V for 1 h. After electrophoresis, the resulting board was illuminated with UV light and photographed with a Bio-Rad ChemDoc XRS. Preparation of ECL sensor. The preparation process of ECL sensor was shown in Scheme 1B. GCE was pretreated by polishing with sand papers of 2000# and 5000# sequentially and then with 0.05 µm alumina powder to obtain a mirror-like surface. Then, GCE was thoroughly rinsed with water and sonicated in ethanol and ultrapure water in turn. Then the GCE was electrodeposited at -2.0 V for 300 s in chitosan (CS) solution to obtain the chitosan modified GCE. 95 µL of CdTe NCs solution (9.3 µM) was first pretreated with 5.0 µL of the mixture of N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide hydrochloride (EDC, 20 mg/mL) and N-hydroxysuccinimide (NHS, 10 mg/mL). The molar ratio of NAC-CdTe NCs to EDC/NHS was controlled strictly to avoid the falloff of some

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Figure 2. (A) Gel electrophoresis image of (lane a) target DNA, (lane b) S1, (lane c) S2, (lane d) S3, (lane e) S4, (lane f) S1S2S3, (lane g) S1S2S4, (lane h) S1S3S4, (lane i) S2S3S4, (lane j) S1S2S3S4, (lane k) target DNA-S1S2S3S4. (B) Cyclic ECL intensity on potential curves of the modified electrode step by step: (a) CdTe NCs/GCE; (b) DNA tetrahedron/CdTe NCs/GCE; (c) BSA/DNA tetrahedron/CdTe NCs/GCE; (d) BSA/DNA tetrahedron/CdTe NCs/GCE after hybridization with 10 pM target DNA and (e) further incubating with Au NDs. Scanning from 0.2 to 1.4 V in 20 mM TPrA (pH 7.4 PBS). Scan rate: 0.1 V s-1. The inset bar graph displayed the ECL intensity of GCE with different modification status from a to e. (C) EIS of the electrode at different stages for (a) bare GCE; (b) CS electrodeposited GCE; (c) CdTe NCs/GCE; (d) DNA tetrahedron/CdTe NCs/GCE; (e) BSA/DNA tetrahedron/CdTe NCs/GCE; (f) BSA/DNA tetrahedron/CdTe NCs/GCE after hybridization with 10 pM target DNA and further incubating with (g) Au NDs and (h) Au NPs. EIS were measured in 0.1 M KCl containing 5 mM [Fe(CN)6]3-/[Fe(CN)6]4-.

ligand groups from the capped NCs surface caused by surplus EDC/NHS, which would subsequently result in the emission disappeared.33 After that the GCE was dipped into the pretreated CdTe NCs solution overnight and then rinsed with water. The CdTe NCs/GCE was immersed in EDC/NHS solution again to activate the exposed carboxyl group modified on the CdTe NCs, in which the ECL intensity did not show obvious changes, suggesting that EDC/NHS for the activation of carboxyl group does not have great influence on the ECL emission of NAC-CdTe NCs immobilized on the surface of GCE (Figure S2). Then the electrode was dipped in 100 µL of the pretreated DNA tetrahedron solution and incubated overnight at 4 °C to graft DNA tetrahedron on the electrode surface via carboxyl-amine attraction (DNA tetrahedron/CdTe NCs/GCE). After rinsing with 20 mM PBS buffer (pH 7.4) containing 5.0 mM Mg2+, the resulting electrode was submerged into BSA (0.2 %) to block the nonspecific active binding sites of CdTe NCs (BSA/DNA tetrahedron/CdTe NCs/GCE), followed by rinsing with 20 mM PBS buffer (pH 7.4) containing 5.0 mM Mg2+ and stored for the further use. Analysis procedure. The modified electrode was immerged into 100 µL of 20 mM PBS buffer containing 5.0 mM Mg2+ and different concentrations of target DNA for 90 min. After washing, the electrode was further dipped into 100 µL of EDC/NHS to activate the carboxyl group located at the top point of DNA tetrahedron. Subsequently, the resulting electrode was incubated with Au NDs at 4 °C for 2 h. After each immobilization step, ECL detection was accomplished with the modified electrode in 4 mL of 0.1 M PBS (pH 7.4) containing 20 mM TPrA and scanned from 0.2 to 1.4 V. Finite-difference time domain (FDTD) calculation. The finite-difference time domain (FDTD) (the package of Lumerical FDTD Solutions 8.15) method was used to perform the simulations about electromagnetic response of gold nanosphere and nanodendrite. The Au nanosphere is modeled as a sphere and Au ND is modeled as a sphere plus with tens of cylinders capped with a half sphere at the end. In the simulations, the refractive index of surrounding medium was set as 1.33. A total-field source with circular polarization by averaging over two orthogonal polarizations was used. And the exci-

tation wavelength was set at 580 nm according to the experiment while mapping the intensity distribution of electromagnetic field on a cross section about single sphere and dendrite.

RESULTS AND DISCUSSION Characterization. In this work, a seeding approach for the growth of spherical gold nanocrystals was applied for the preparation of Au NDs in which 25 nm Au NPs were used as the seeds and a mixture HAuCl4 and hydroxylamine as growth solution.1 The transmission electron microscopy (TEM) image shows that MCH and cystamine-modified Au NDs possessed a uniform diameter size of 60 nm (Figure 1A). Furthermore, each Au ND had tens of elongated sharp subunits and the long size of subunit was about 10 nm (Figure 1B). In Figure 1D, TEM image of CdTe NCs showed that the average size of synthesized CdTe NCs was about 3.0 ± 0.4 nm. As the UV-vis absorbance spectra showed (Figure 1E), the NAC modified CdTe NCs have a broad range of absorption with the first excitonic absorption peak located at ca. 500 nm, according to which the diameter was calculated to be 2.8 nm in theory.34 And this result was consistent with the TEM image. Also a great fluorescence signal was obtained with the wavelength centered at 540 nm. To demonstrate the successful formation of DNA tetrahedron after annealing and the response feasibility of hairpin structure to target DNA, agarose gel electrophoresis was carried out and shown in Figure 2A. Lanes a to e showed the single strand DNA bands of target DNA, S1, S2, S3 and S4, respectively. Bands of three strands DNA combinations (S1S2S3, S1S2S4, S1S3S4, S2S3S4) could be observed in lanes f to i, which showed larger molecular weight than single strand DNA. Moreover, the band of DNA tetrahedron (lane j, prepared by S1S2S3S4) shifted slower than the single strand DNA bands (lanes a-e) and the combination bands of three strands (lanes f-i), indicating the successful formation of hairpin structure DNA tetrahedron. Compared with linear or more compact structures, the increased mass and spatial complexity of DNA tetrahedral scaffold hindered the movements through the pores of hydrogel.35 After adding target DNA in the hairpin structure DNA tetrahedron, the band of S1S2S3S4-target

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Figure 3. (A) UV-vis absorption spectra of a) Au NPs and b) Au NDs under different modification states: before (curve a), after incubated with cystamine in bulk solution with time elapse (from curve b to d) and asymmetrically modified with MCH and cystamine (curve e), inset shows the picture of Au NPs and NDs during the modification process; Bar graph of ∆ECL intensity (I − I0) of sensors in response to Au NDs with (blue bars) and without (yellow bars) asymmetric modification, and the length of DNA tetrahedron was set as 6 nm (B and D) and 12 nm (C and E) respectively.

DNA (lane k) shifted slower than the band of S1S2S3S4 (lane j), suggesting the successful responses of hairpin structure to target DNA. The ECL responses of the sensor after each fabricated procedure were recorded in Figure 2B. When chitosan electrodeposited GCE was modified with CdTe NCs, a distinguished ECL peak appeared (curve a). The CdTe NCs acted as ECL emitter, and generated an ECL peak at ca. 1.38 V in the presence of coreactant TPrA. In this anodic process, CdTe was oxidized to CdTe•+ while TPrA was oxidized to TPrA•. After that, CdTe•+ can react with TPrA• and reached to excited state CdTe*. While returned to ground state, the CdTe emit light and strong ECL signal can be obtained.36 After the assemblies of DNA tetrahedron (curve b) and BSA (curve c), the ECL intensities decreased gradually because of the formation of less conductive layers on the electrode surface. While, after further incubation of target DNA, a slightly enhanced ECL signal was obtained (curve d). This phenomenon could be attributed to the reconfiguration of DNA tetrahedron, resulting in a nanospacing enhancement on the electrode surface. The hollow structures of the tetrahedron could reduce the surface effect and increase the layer thickness of solution-phaseenvironment.37 Subsequently, with the inclusion of Au NDs onto the top vertex of DNA tetrahedron, the ECL signal was improved considerably. The ECL enhancement was mainly caused by the presence of strong electromagnetic field resulting from plasmon coupling effect between multiple branches of Au NDs. Electrochemical impedance spectroscopy (EIS) is a powerful and sensitive technique for charactering the changes of interfacial properties in the stepwise assembly processes of electrode surface modification.38 In Figure 2C, [Fe(CN)6]3-/4redox couple were used to monitor and conform the construction process of the biosensor. Compared with bare GCE (curve a, Ret = 105 Ω), an increased semicircle diameter was ob-

tained for CS modified GCE (curve b, Ret = 2100 Ω) and CdTe NCs/GCE (curve c, Ret = 2261 Ω). After the selfassemblies of less conductive DNA tetrahedron (curve d, Ret = 2670 Ω) and BSA (curve e, Ret = 2930 Ω), electron transfer impedance Ret increased gradually. In the presence of target DNA, hybridization to complementary DNA resulted in a slight decrease of Ret (curve f, Ret = 2839 Ω), agreeing with the aforementioned ECL characterization of tetrahedral biosensor (Figure 2B, curve d). Finally, when Au NDs or Au NPs were assembled to the top of DNA tetrahedron, a much lower resistance was obtained at around 1510 Ω (curve g) for Au NDs and 1566 Ω (curve h) for Au NPs, leading to an enhancement in the electron transfer kinetics of [Fe(CN)6]3-/4-. As curve g and h depicted, Au NDs and NPs contributed similarly to enhance the efficiency of electron transfer. Overall, ECL and EIS measurements all demonstrated the successful fabrication of the DNA biosensor. ECL Enhancing Mechanism of CdTe NCs from Au NDs. DNA nano-scaffolds with exterior modification (encoding a hairpin structure into one component DNA strand) were grafted on the surface of CdTe NCs/GCE via amino linkers at 5’ ends of their three component strands (S2, S3, S4). At this moment, DNA tetrahedron was at relaxed state and the carboxyl group attached on the top vertex of DNA tetrahedron was at close proximity (4.8 nm) to the electrode surface. After treatment of EDC/NHS, amine-terminated Au NDs were directly assembled on the top vertex of DNA tetrahedron by amine-carboxyl covalent bonding39, bringing in the ECL intensity quenching of CdTe NCs due to FRET effect, which was named as “turn off” mode. The asymmetric modification made Au NDs have the available top point modified with amine group and achieve the fixed distance between Au NDs and CdTe NCs, bringing in a better reproducibility (Figure 3B and C). The relative standard deviation (RSD) was around 2.3 % for 10 pM target DNA using eight different tests, indicating

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Figure 4. (A) The FDTD simulation of electric field intensity (|E|2) for single Au nanodendrite (a) and sphere (b). ECL signals of sen-sors before (curve a) and after (curve b) incubating with Au NDs (B and C) and Au NPs (D and E), in which the length of DNA tetrahedron was set as 6 nm (B and D) and 12 nm (C and E) respectively.

acceptable fabrication reproducibility. In Figure 3D and E, BSA/DNA tetrahedron with 6 nm or 12 nm edge length/CdTe NCs/GCE incubating with Au NDs without any asymmetric modification possessed of poor reproducibility. Due to the spherical structure, Au NPs without asymmetric modification still have good reproducibility (Figure S3). Hence, the asymmetric modification for irregular dendritic structure of Au NDs was indeed necessary which help to increase the repeatability and stability of sensor. In addition, the asymmetric modification method ensured the stability of amine modified Au nanoparticles. As Figure 3A depicted, the UV-vis absorption peak of MCH and cysteamine asymmetrically modified Au NPs and NDs (curve e) red shifted for about 10 nm compared with pure Au NPs and Au NDs (curve a). However, when cysteamine modification was conducted in bulk solution without asymmetric method, the intensity of UV-vis absorption decreased and the peak became broader with red shifting (curve b to d). Au NPs and NDs in solution were unstable and tend to aggregate then precipitate after minutes (inset picture in Figure 3A), which again certified the importance and necessity of asymmetric modification method. Hybridization to complementary target DNA resulted in a significantly increased ECL signal, consistent with switching of the tetrahedral configuration from the hairpin closed state to the hybridized extended structure. Based on 0.34 nm for one base pair,23 tetrahedron scaffold with 12 nm edge length shows 9.8 nm in height. This reconfiguration was responsible for the increased distance between the Au NDs and CdTe NCs surface. Therefore, due to the energy transfer of ECL excited LSPRs between multiple tip branches of Au NDs to CdTe NCs at large separation, 6.1-fold ECL intensity enhancement (Figure 2B, defined as I/I0, where I and I0 are the ECL intensities of target DNA/BSA/DNA tetrahedron/CdTe NCs/GCE in the presence and absence of Au NDs, respectively) was achieved after hybridization with 10 pM target DNA. In order to prove that the availability of quenching and enhancement of ECL signals from CdTe NCs by Au NDs relied on their distance,

taut DNA tetrahedron with 6 nm or 12 nm edge length were investigated in Figure 4. When the edge length of DNA tetrahedron was 6 nm with the 4.8 nm in height, the ECL signal of CdTe NCs was quenched by the close proximity of Au NDs (Figure 4B). However, after the incubation of DNA tetrahedron with 12 nm edge length in 9.8 nm height, the ECL intensity from CdTe NCs got 6.1-fold enhancement by the plasmon effect of Au NDs at large separation (Figure 4C), which also verified the mechanism aforementioned. As non-spherical noble metal nanoparticles, Au NDs possessed some distinctive topological and morphological features, such as well-defined superstructure, moderately elongated subunits and sharp tips and edges.2,9 Due to the plasmon located around the branches of Au NDs, a strong electromagnetic field could be obtained, which could excite the optical behavior of CdTe NCs and lead to the occurring of an ECL enhancement. To prove the unique strengths of sharp tips from Au NDs, gold nanoparticles (Au NPs) with an approximate 60 nm diameter were used to replace Au NDs with the same modification process. TEM showed that Au NPs modified with MCH and cysteamine possessed good uniformity (Figure 1C). After the BSA/DNA tetrahedron with 6 nm edge length/CdTe NCs/GCE incubating with Au NPs, a quenching of ECL intensity was obtained (Figure 4D). Compared with 60 nm Au NPs with 551 nm absorption peak in UV-vis spectrum after asymmetric modification, Au NDs with around 585 nm absorption peak showed more overlap degree with the ECL emission of CdTe NCs at 585 nm (Figure 1F), which was a key element for efficient energy transfer in ECL systems.36,40 When the BSA/DNA tetrahedron with 12 nm edge length/CdTe NCs/GCE was further incubated with Au NPs with 60 nm diameter, only 1.7-fold ECL enhancement was obtained (Figure 4E). As Figure 4A depicted, due to the plasmon coupling of many subunits from Au NDs, very high electric field intensity (“hot spot” effect) was gained, which was higher than that of the spherical Au NPs. Such a plasmon coupling effect can also effectively boost up the rate of excitation, resulting in

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higher ECL signal enhancement.9 Furthermore, the optical property of nanodendrites with different sizes and materials were evaluated with the assistance of FDTD simulation. As displayed in Figure S4, for Au NDs with diameter of 30 nm, the absorption peak blue shifted for 20 nm, resulting in a lower spectra overlap efficiency with the ECL emission centered at 585 nm. Also, the electric field intensity (|E|2) was five times lower than that of Au NDs with diameter of 60 nm. For Ag NDs with diameter of 60 nm, the absorption peak blue shifted for more than 100 nm, which did not match with the ECL emission of CdTe NCs. Besides, while in positive potential scanning, the silver nanoparticles were unstable and tend to be dissolved41. In view of the above-mentioned factors, the Au NDs with diameter of 60 nm were more suitable and were finally chosen as plasmons to conduct the assay of target nucleic acid.

Figure 5. (A) ECL-time curves on the target DNA with various concentrations (from a to n: 0 to 10 pM) (B) Dependence of ∆ECL intensity of CdTe NCs on the concentration of target DNA. Inset: linear relationship between ∆ECL intensity and the concentration of target DNA. (C) The excellent stability of plasmon mode-induced enhancement of ECL from CdTe NCs. (D) Bar graph of ∆ECL intensity for BSA/DNA tetrahedron/CdTe NCs/GCE after being incubated with various DNA sequences and Au NDs in sequence. Various DNA sequences were followed as (a) no target DNA, (b) single-base mismatched DNA sequence, (c) three-base mismatched DNA sequence, (d) non-complementary DNA sequence, (e) complementary target DNA and (f) the mixture of above sequences respectively. Five measurements were performed for each point. ECL measures were carried out in 0.1 M PBS (pH 7.4) containing 20 mM TPrA. Voltage of the photomultiplier tube was set at 700 V and scan rate was 100 mV s-1.

Analytical Performance. To evaluate the potential quantitative application and the sensitivity of the LSPR enhanced ECL biosensor, various concentrations of target DNA were assayed. Figure 5A was the ECL signal-time curves which showed the relationship between the ECL intensity and the concentration of target DNA from 0 to 10 pM. As shown in Figure 5B, ∆ECL intensity of CdTe NCs was increased with the increase of the concentration of target DNA. From the inset of Figure 5B, it can be observed that ∆ECL intensities was linearly proportional to the target DNA concentration in the range of 1.0 fM to 500 fM with a correlation coefficient (R) of 0.997. The detection limit was 30 aM at a signal-to-

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noise ratio of 3. In Table S2, this proposed strategy provided higher sensitivity and lower detection limitation compared with other reported methods. The ECL stability of this plasmon-based ECL biosensor was also examined by monitoring of its ECL response toward 10 pM target DNA under consecutive cyclic potential scans for 20 cycles. As described in Figure 5C, the ECL responses did not show any obvious changes. In addition, the selectivity of the plasmon-based ECL biosensor was further studied by measuring the ECL responses to different types of DNA sequences (10 pM) including completely complementary target DNA sequence, single-base mismatched DNA sequence, three-base mismatched DNA sequence, non-complementary sequence and the mixture of those sequences, respectively. As shown in Figure 5D, when no target DNA exist, the ∆ECL intensity was about -1460 a.u. (bar a). In the presence of single-base (bar b) mismatched sequence, ∆ECL intensity was a little bit increased, but still much lower than that of complementary target DNA sequence (bar e). The three-base (bar c) mismatched sequences and noncomplementary sequence (bar d) almost induced no changes. And the response to the mixture of above sequences was nearly the same as complementary sequences. Such results indicated that the developed plasmon-based ECL sensing system could exhibit an acceptable sequence specificity and selectivity for multiplex detection. Besides, to evaluate the practical application of this plasmon-based ECL biosensor, recovery testing was also examined by spiking target DNA solution into 10 % human serum (Table S3). The spiked sample with different concentrations of target DNA was measured by the proposed method. As displayed in Table S3, the recoveries varied from 98% to 106%. The results demonstrated that the proposed ECL biosensor for DNA detection could be applied in real sample analysis.

CONCLUSION In this work, we demonstrated an LSPR-induced enhancement of ECL emission from CdTe NCs based on the strong electromagnetic field effect located around the branch of Au NDs. Due to its mechanical reconfiguration upon target DNA hybridization, DNA tetrahedron self-assembled onto electrode surface underwent the ECL addressable nanoswitching. Moreover, this controllable re-configurational switching of the surface-confined tetrahedral structures along with controllable asymmetric functionalization of Au NDs in response to target DNA took place with a good efficiency. We believe that this study could broaden the perspective of non-spherical noble metal nanoparticles for further development of ECL biosensors.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Reagents and apparatus, Zeta potential of Au NDs and Au NPs, the influence of EDC/NHS treatment on CdTe NCs, ECL response of sensor induced by Au NPs with different modification method, the FDTD simulation of nanodendrites with different parameters. (PDF)

AUTHOR INFORMATION

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Analytical Chemistry

Corresponding Author *Tel/Fax: +86-25-83597294. E-mail: [email protected]. *Tel/Fax: +86-25-83597294. E-mail: [email protected]

ORCID Jing-Juan Xu: 0000-0001-9579-9318 Author Contributions †M.-X. L, and Q.-M. F. contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work was supported by National Key R&D Program of China (Grant 2016YFA0201200), the National Natural Science Foundation of China (Grant Nos. 21475058 and 21535003). This work was also supported by a Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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