Self-Assembled DNA Tetrahedral Scaffolds for the Construction of

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Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification Qiu-Mei Feng, Yue-Hua Guo, Jing-Juan Xu, and Hong-Yuan Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 04 May 2017 Downloaded from http://pubs.acs.org on May 5, 2017

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ACS Applied Materials & Interfaces

Self-Assembled DNA Tetrahedral Scaffolds for the Construction of Electrochemiluminescence Biosensor with Programmable DNA Cyclic Amplification †‡

Qiu-Mei Feng, †

‡

Yue-Hua Guo, Jing-Juan Xu,‡* and Hong-Yuan Chen‡

School of Chemistry and Chemical Engineering, Jiangsu Normal University, Xuzhou

221116, P. R. China ‡

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, P. R. China

KEYWORDS: electrochemiluminescence (ECL), DNA tetrahedron, programmable DNA cyclic amplification, multilayers, biosensors

______________________________ * Corresponding author. Phone/Fax: +86-25-89687294. E-mail: [email protected] 1

ACS Paragon Plus Environment

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

ABSTRACT: A novel DNA tetrahedron-structured electrochemiluminescence (ECL) platform for bioanalysis with programmable DNA cyclic amplification was developed. In this work, glucose oxidase (GOD) was labelled to a DNA sequence (S) as functional conjugation (GOD-S), which could hybridize with other DNA sequences (L and P) to form GOD-S:L:P probe. In the presence of target DNA and a help DNA (A), the programmable DNA cyclic amplification was activated and released GOD-S via toehold-mediated strand displacement. Then, the obtained GOD-S was further immobilized on the DNA tetrahedral scaffolds with a pendant capture DNA and Ru(bpy)32+-conjugated silica nanoparticles (RuSi NPs) decorated on the electrode surface. Thus, the amount of GOD-S assembled on the electrode surface depended on the concentration of target DNA and GOD could catalyze glucose to generate H2O2 in situ. The ECL signal of Ru(bpy)32+-TPrA system was quenched by the presence of H2O2. By integrating the programmable DNA cyclic amplification and in situ generating H2O2 as Ru(bpy)32+ ECL quencher, a sensitive DNA tetrahedron-structured ECL sensing platform was proposed for DNA detection. Under optimized conditions, this biosensor showed a wide linear range from 100 aM to 10 pM with a detection limit of 40 aM, indicating a promising application in DNA analysis. Furthermore, by labelling GOD to different recognition elements, the proposed strategy could be used for the detection of various targets. Thus, this programmable cascade amplification strategy not only retains the high selectivity, good capturing efficiency of tetrahedral-decorated electrode surface, but also provides potential applications in the construction of ECL biosensor.

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■ INTRODUCTION In recent years, to realize a sensitive detection for targets, nanostructured electrode surface was designed to enhance the accessibility of target molecules or beacon materials in the construction of biosensors.1-4 For example, Fan’s group grafted DNA tetrahedral scaffolds on the electrode surface to construct electrochemical sensors for the detection of prostate-specific antigen,5 cocaine,6 microRNA7 and telomerase activity from cancer cells,8 demonstrating stronger electrochemistry sensitivity than double stranded DNAs based sensor. Due to the mechanical rigidity, structural stability, high precision, and ease of preparation, DNA tetrahedron could reduce the local overcrowding effect with well-defined spacing and enhance spatial position range, leading to higher capturing efficiency.5,9,10 Moreover, DNA tetrahedron with three-dimensional scaffold exactly controlled recognition units and easily functioned with different chemical biomolecules or moieties, such as fluorescence molecules,11,12 metal nanoparticles,13-15 DNA probes,16-18 and so on. Recently, our group designed an electrochemiluminescence (ECL) sensor based on DNA tetrahedral scaffolds inserted with telomerase strand primer for the determination of telomerase activity, bringing in the excellent controllability, high precision, enhanced spatial dimension and low detection limit.10 To further improve the measuring sensitivity for trace biological samples, seeking for a kind of novel DNA tetrahedral nanostructured ECL sensor coupling with signal amplifying strategy was promising. Benefitting from molecular programming, non-enzymatic DNA circuits for amplifying signals by governing the simple rules of nucleic acid hybridization have been proposed and drawn particular interest in sensing. Various enzyme-free DNA amplification strategies based on

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molecular programming such as hybridization chain reaction,19,20 the catalyzed hairpin assembly21-23 and entropy-driven catalysis24,25 have been used for the development of low-cost point-of-care diagnostics. Among these, the programmable entropy-driven catalysis was one of the most promising methods due to the unique and exclusive entropy-driven force.24-27 For example, combined with fluorescent monitoring or optical whispering gallery mode resonance, the entropy-driven enzyme-free DNA amplification system was programmed to determine DNA, resisting disturbance from a complex environment and bringing in a low detection limit.26,27 Taking into account the above advantages of DNA tetrahedron and programmable DNA cyclic amplification, a novel recycling amplified ECL biosensor was designed on the basis of DNA tetrahedral scaffolds modified platform. In this approach, the multilayers of {RuSi NPs} were constructed on the electrode surface to boost ECL emission. Then, one fragment was appended to the rigid DNA tetrahedron as capture DNA. This bulky tetrahedral structure grafted electrode surfaces not only avoided inter-capture DNA entanglement via spatially segregating pendant strands, but also placed capture DNA in a solution-phase-like environment, resulting in the improvement of hybridization efficiency for glucose oxidase (GOD)-conjugated DNA sequence. In programmable DNA cyclic system, target DNA as an input sequence could catalyze the release of GOD-conjugated DNA sequence, which was then catalyze glucose to generate H2O2. Thus, the increasing H2O2 led to a sensitive ECL signal change of Ru(bpy)32+-TPrA system for target DNA detection.

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■ EXPERIMENTAL SECTION

Materials and Reagents. All the DNA oliogonucleotides were synthesized and purified using high performance liquid chromatography by Sangon Biological Engineering Technology & Services Co. Ltd (Shanghai, China) and suspended in deionized water to be a final concentration of

100

µM.

The

corresponding

DNA

sequences

were

listed

in

Table

S1.

N-(3-dimethylaminopropyl)-N-ethyl-carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), p-aminobenzoic acid (ABA), ethylenediamine (En), NH3·H2O, bovine serum albumin (BSA), glutaraldehyde (GA), tris (2, 2-bipyridyl) dichlororuthenium (Ⅱ) hexahydrate, triton X-100, 1-hexanol, cyclohexane, tetraethyl orthosilicate (TEOS), acetone, (3-aminopropyl) triethoxysilane

(APTES),

tris(2-carboxyethyl)

phosphine

hydrochloride

(TCEP),

maleimidobenzoic acid N-hydroxy-succinimide ester (MBS), glucose oxidase (GOD), dimethyl sulphoxide (DMSO) and tripropylamine (TPrA) were all obtained from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. H2O2 (analytical reagent grade) and D-(+)-glucose was from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The ECL detection solution comprised of 30 mM TPrA and 10 mM glucose were prepared in 0.1 M phosphate buffer solution (PBS, pH 7.4). The Millipore ultrapure water with 18.2 MΩ. cm resistivity was used throughout the experiment. The other chemical reagents were all analytical reagent grade and applied as received. Apparatus. ECL emission measurements and cyclic voltammetry were monitored, respectively, with a MPI-A multifunctional ECL analytical system (Xi’an Remax Electronic Science & Technology Co. Ltd., Xi’an, China) and a CHI 660C electrochemical workstation 5



(Shanghai, China). For ECL experiments, the voltage of photomultipliter tube (PMT) was set at -800 V and the potential scan was from 0 to 1.40 V with 100 mV s-1 scan rate. The experiment was performed on conventional three-electrode system comprising a glassy carbon electrode (GCE) as working electrode, a platinum wire counter electrode and saturated calomel electrode (SCE) reference electrode. Electrochemical impedance spectroscopy (EIS) experiments were acquired with an Autolab PGSTAT30/FRA2 system (Netherlands). To verify the formation of TN, GOD-S and the feasibility of programmable DNA cyclic amplification, polyacrylamide gel electrophoresis (PAGE) analysis was carried out by using a Bio-Rad electrophoresis analyzer (USA) and imaged on the Bio-Rad ChemDoc XRS (USA). For characterization of synthetic RuSi NPs, a Hitachi S-3000N scanning electron microscope (SEM, Japan) was utilized. Synthesis and Modification of RuSi NPs. RuSi NPs with the average diameter of 60 nm were prepared according to the previous work with a slight modification.28 First, 7.5 mL of cyclohexane, 1.77 mL of Triton-X-100 and 1.8 mL of 1-hexanol were mixed with constant magnetic stirring for 15 min, then 340 µL of 40 mM Ru(bpy)32+ was added and stirred for 30 min, forming a water-in-oil microemulsion. After the addition of 60 µL of NH3·H2O and 100 µL of TEOS to the above solution, the stirring reaction was kept constant for 24 h at room temperature. Then, acetone was slowly injected to the above solution and the emulsion was destroyed. The final reaction solution was centrifuged to collect precipitates, which were washed thoroughly with ethanol and water for three times, respectively. After that, 10% APTES solution (prepared by ethanol) was added to orange-colored RuSi NPs and incubated for 2 h, bringing in the formation of amino group functionalized RuSi NPs. Finally, the obtained nanoparticles were

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carefully washed with ethanol to remove loosely bound APTES and then dispersed in 0.1 M PBS to employ for the assembly of ECL emitter on the surface of electrode. Conjugation of GOD to DNA. MBS, as a bifunctional linker,29 can be used to prepare the conjugation of 5’-SH-modified S strand to GOD (named as GOD-S). At the beginning, 25 µL DMSO solution containing 6.4 mM MBS was added in 1 mL of 1 mg/mL GOD. After vortexing for 5 min, the mixed solution was shook at room temperature for 1 h, resulting in maleimidobenzoic GOD amide. Then, the obtained solution was purified with Millipore (100 kDa) to remove excess MBS. Finally, MBS-activated GOD was reacted with 1 mL of 100 µM thiolated S strand and the conjugating product was further purified with Millipore (100 kDa), resuspending in 500 µL PBS. Before use, thiolated S strand needed to be activated by 10 mM TCEP for 1 h, in order to reduce disulfide bonds. Fabrication of DNA tetrahedron-structured electrode surface. The fabrication of DNA tetrahedron-structured electrode surface was illustrated in Scheme 1. Firstly, GCE was successively polished in sequential order with 1.0, 0.3 and 0.05 µm of alumina power to make the electrode surface mirrorlike. Subsequently, for grafting amino group functionalized RuSi NPs, an assembly of ABA was formed on GCE surface (ABA/GCE) through carbon-nitrogen linkage under cyclic voltammetry from 0.40 to 1.20 V in pH 7.4 PBS containing 1.0 mM ABA at 10 mV s-1 for four cycles.30 After washing with ethanol and dried with N2, ABA/GCE was immersed into PBS containing 20.0 mg mL-1 EDC, 10.0 mg mL-1 NHS and 2.0 % (v/v) En at the room temperature for 1 h to form En/ABA/GCE, followed by rinsing with PBS. Then, En/ABA/GCE was immersed into 2.5% (v/v) GA solution for 2 h at 37 °C and conjugated with

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amino group functionalized RuSi NPs to obtain {RuSi NPs}/En/ABA/GCE. The first {RuSi NPs} layer modified En/ABA/GCE was further immersed into GA solution and RuSi NPs to form {RuSi NPs}2/En/ABA/GCE. By repeating the dipping procedures in the above two different solutions for four times, the multilayers of {RuSi NPs}4 on the electrode surface (named as {RuSi NPs}4/En/ABA/GCE) can be obtained. DNA tetrahedral nanostructure (TN) was synthesized according to the literature with slight changes.31 Equimolar amounts of four constituent oligonucleotides (S1, S2, S3 and S4, the sequences in Table S1) were mixed in 20 mM PBS buffer (containing 5.0 mM Mg2+, pH 8.0) with a final concentration of 1 µM. Then, this mixture was placed at 95 °C for 5 min, and cooled to the ambient temperature. Finally, the obtained TN solution was purified by 30 K molecular weight cutoff ultrafiltration to remove the non-conjugated oligonucleotides. TN contained one pendant DNA segment as capture DNA at one vertex and three amine groups at the other vertices. The side length of the obtained TN was 6.12 nm based on 0.34 nm for one base pair. Next, {RuSi NPs}4/En/ABA/GCE was immersed into GA solution for 2 h at 37 °C and then incubated with TN solution to graft TN on the electrode surface (named as TN/{RuSi NPs}4/En/ABA/GCE). After washing with PBS, 2% BSA was used for 1 h to block the unoccupied sites of GA, followed by rinsing with PBS and achieved BSA/TN/{RuSi NPs}4/En/ABA/GCE for the further use. Gel electrophoresis. The formation of TN in solution was confirmed through PAGE. Different DNA structures (S1, S2, S3, S4, S1S2S3, S1S2S4, S1S3S4, S2S3S4 and S1S2S3S4) were all placed at 95 °C for 5 min, cooling to ambient temperature. Then, the gel electrophoresis

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was performed by injecting the loading samples (the mixture of different DNA structures, UltraPower TM dye and loading buffer) into a 5 % native polyacrylamide gel. The loading samples were placed for 3 min and then injected into polyacrylamide hydrogel. The electrophoresis analysis was carried out at 100 V for 1.5 h and then the resulting board was observed under UV irradiation. Finally, Molecular Imager Gel Doc XR was applied to photograph. Besides, 10 % native polyacrylamide gel was used to assay the GOD-S conjugation as the above procedure. After electrophoresis, the resulting gel board can be further used for coomassie brilliant blue analysis. Briefly, the gel was stained in 0.025 % (w/v) coomassie brilliant blue R-250 dye for 1 h and then destained in a solution containing 5 % methanol and 7 % acetic acid for 5 h at room temperature. Analysis procedure. For the fabrication of GOD-S, all the applied DNA strands were annealed by heating at 95 °C for 5 min and then slowly cooled down to room temperature before use. First, GOD-S:L:P probe was obtained by hybridizing of L, P and GOD-S in equimolar ratio at room temperature for 1 h with a final concentration of 0.5 µM. Next, 0.5 µM assistant DNA (A) was added into the probe solution and then different concentrations of target DNA was added, incubating at room temperature for 1 h. Then, BSA/TN/{RuSi NPs}4/En/ABA/GCE was incubated with the above solution for 1 h at 37 °C. After washing with PBS buffer containing 5.0 mM Mg2+ thoroughly, bringing GOD-S to the modified electrode surface. ECL detection was accomplished with the modified electrodes in 0.1 M PBS (pH 7.4) containing 30 mM TPrA and 10 mM glucose.

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Scheme 1. Schematic illustration of the programmable DNA cyclic amplifying ECL sensor for the detection of DNA via DNA tetrahedron-structured platform.

■ RESULTS AND DISCUSSION

Characterization. As an ECL luminophore, due to its high ECL efficiency and good stability in aqueous solution, Ru(bpy)32+ and its derivatives have been the most extensively studied luminophores.32 In this work, a large amount of Ru(bpy)32+ molecules were encapsulated in silica nanoparticles (abbreviated as RuSi NPs) to greatly enhance the ECL signal. In Figure 1A, the scanning electron microscopy (SEM) image showed a homogeneous distribution of around 60 nm in diameter for RuSi NPs. To confirm the correct formation of TN, PAGE was carried out. In Figure S1, compared with the combinations of three strands (lanes 2-5) and the single strand DNA (lanes 6-9), a new band

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of lower mobility appeared for TN (lane 1). For TN, the increased mass and spatial complexity both hindered the movements through the polyacrylamine hydrogel. Then, PAGE was further used to verify programmable DNA cyclic cascade amplification. As shown in Figure 1B, lane 4 was the band of L:P:S complex with lower migration rate than the incubating single strand S, P and L (lanes 1-3). After L (lane 3) and A (lane 5) incubating at room temperature for 1 h, a new band appeared in lane 6, indicating the formation of L:A duplex. When L, P and target DNA incubated together, a new band appeared in lane 7, indicating the formation of L:P:target DNA complex. For L:P:S complex, in the presence of target DNA, the obtained band (lane 8) was at the same position as that of L:P:target DNA complex (lane 7), indicating that target DNA could displace S from the L:P:S complex. In addition, a weak band in lane 9 was observed when A was added in L:P:S complex, displaying the formation of L:A duplex with little amount. However, when target DNA was incubated with the mixture of L:P:S complex and A, according to lane 6, the bands of L:A duplex was obvious (lanes 10 and 11). Meanwhile, the band of L:P:S complex in lane 10 disappeared and in lane 11 was inapparent, but target DNA added in lane 11 was ten less than lane 10, which indicated the amplifying feasibility of programmable DNA cascade assembly reaction. Besides, PAGE and protein-stained image were both used to verify the formation of GOD-S conjugation. As displayed in Figure 1C, the band in lane 2 and lane 3 was the thiolated-S strand and its mixture with GOD, respectively. The very weak band was the trace disulfide. Upon the addition of MBS to the mixture, a new band was stuck in the wells (lane 4), resulting from the large molecular weights of the purified GOD-S conjugates. Compared with lane 2, the band of S

11



strand disappeared (lane 4), which also suggested that unreacted DNA was efficiently removed from the product mixture by centrifugation. Then, protein-stained image with coomassie on the same gel for the detection of GOD on the conjugation products was operated. As shown in Figure 1D, no difference was observed among GOD (lane 1), the mixture of S and GOD (lane 3) and the conjugate of GOD-S (lane 4). The molecular weight of GOD was too large for migration in PAGE even if the GOD was conjugated to DNA. The band of S in lane 4 of Figure 1C and the band of GOD in lane 4 of Figure 1D was at the same position, which also indicated the successful coupling of S to GOD.

Figure 1. (A) SEM image of RuSi NPs. (B) Gel electrophoresis analysis image of programmable DNA cyclic amplification. Lane 1: S, lane 2: P, lane 3: L, lane 4: L:P:S complex, lane 5: A, lane 6: L:A duplex, lane 7: L:P:target complex, lane 8: the mixture of L:P:S complex and target DNA, lane 9: the mixture of L:P:S complex and A, lanes 10-11: the mixture of L:P:S 12


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complex, A and target DNA. The concentrations of sequences were all 10 µM, except target DNA used in lane 11 was 1 µM. (C) Gel electrophoresis image and (D) protein-staining image of the conjugation of GOD to S. Lane 1: GOD, lane 2: thiolated-S, lane 3: the mixture of thiolated-S and GOD, lane 4: reaction product of S with GOD in presence of MBS (GOD-S).

Construction and characterization of the ECL biosensor. In the presence of coreactant TPrA, as the electrode potential was sufficiently positive, Ru(bpy)32+ could be oxidized to form a strong oxidant of Ru(bpy)33+, and TPrA could be simultaneously oxidized to cation radical TPrA+˙. TPrA+˙could rapidly form TPrA˙ by losing a proton. TPrA˙ as a strong reductant could react with Ru(bpy)33+ to achieve an excited state Ru(bpy)32+*, producing an ECL signal. In this Ru(bpy)32+-TPrA ECL system, after the addition of H2O2, radical TPrA˙ could be annihilated by H2O2 or the radical produced during the oxidation of H2O2, bringing in the decrease of ECL signal.33,34 Figure 2A displayed the influence of the thickness of {RuSi NPs}n films on the ECL signals. With the increase of {RuSi NPs} layer number from 1 to 6, ECL intensity gradually increased, reached the maximum at the number of 4 and then decreased, because larger number of {RuSi NPs} layer assembled on the electrode surface also blocked electron transfer. Therefore, {RuSi NPs}4 films were selected in the following experiments. In Figure 2B, {RuSi NPs}4 films modified En/ABA/GCE was utilized to measure different concentrations of H2O2 on the basis of the significant quenching effect of H2O2 on the Ru(bpy)32+ ECL. With an increase of H2O2 concentration from 0 to 100 pM, the ECL intensity markedly decreased. These results illustrated that H2O2, which was produced from the oxidase reaction, can be quantitatively detected. Thus, a programmable cascade signal amplifying ECL sensor coupled with DNA tetrahedral 13



scaffolds-based platform was constructed by the introduction of GOD on the electrode surface for the indirect determination of target DNA. As illustrated in Scheme 1, P strand and GOD-modified S strand can hybridize with the linker strand (L) to form three-stranded substrate complex (L:P:S) complex. Though A was complementary to L strand in the complex, no significant reaction occurred, and the two species were metastable. In the presence of target DNA, the GOD-modified S strand could be displaced and formed L:P:target complex. A strand could hybridize with L strand in the L:P:target complex, resulting in L:P:A:target intermediate, which then quickly rearranges to release target DNA and P strand and generate duplex L:A. At the end of cascade assembly reaction, P was released as a single-stranded product, and target was released to enable multiple turnover. To enhance ECL emitting, the multilayers of {RuSi NPs} were assembled on the electrode surface. Then, TN modified with amino groups at three vertices was attached on the GA treated RuSi NPs surface and a segment DNA linker as capture DNA was placed at the top vertex of DNA tetrahedron. DNA tetrahedral scaffolds could control the nanospacing between capture DNA, providing higher accessibility and controllability for the assembly of GOD-S. Thus, GOD was attached to the electrode surface by the complementary hybridization between TN and S strand. GOD could catalyze glucose to generate H2O2 in situ and the ECL signals of Ru(bpy)32+ was quenched.

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Figure 2. (A) The ECL intensity of {RuSi NPs}n film assembled on GCE surface: n = 1-6. (B) ECL curves for {RuSi NPs}4/En/ABA/GCE in pH 7.4 PBS solution containing different concentrations of H2O2 (from a to e: 0, 100 fM, 1 pM, 10 pM, 100 pM).

As a facile and powerful electrochemical technique to probe the changes of interfacial properties of electrodes, EIS was utilized to monitor the assembly process.35 As shown in Figure 3A, for the bare GCE, the impedance spectrum exhibited a small semicircle (curve a, Ret = 280 Ω). After ABA electrodepositing on the electrode surface, because of the electrostatic repulsion between ferricyanide anions and negatively charged ABA/GCE surface (pKa-COOH of ABA, 4.80), the resistance Ret increased obviously (curve b). The decreased Ret of En/ABA/GCE (curve c, Ret = 2232 Ω) to that of ABA/GCE (curve b) should due to the electrostatic attraction between ferricyanide anions and the positively charged En/ABA/GCE surface (pKa-NH2 of En, 9.93). When {RuSi NP}4 was immobilized on the surface of En/ABA/GCE, the Ret increased to 6600 Ω (curve d), ascribing to the formation of less conductive layers on the electrode surface. Then, the self-assembly of TN (curve e, Ret = 8270 Ω), BSA (curve f, Ret = 10490 Ω) and the following GOD-S (curve g, Ret = 13380 Ω) all blocked the transfer of electrons to the electrode surface, 15



thus dramatically increased the impedance value. Besides, the ECL signals were also applied to characterize the fabrication process of the sensing system in Figure 3B. When {RuSi NP}4 films was constructed on En/ABA/GCE surface, an outstanding ECL signal was achieved (curve a). Subsequently, with the grafting of TN (curve b) and BSA (curve c), the ECL signals both decreased. After the incubation of GOD-S, ECL intensity further obviously decreased, which was attributed to the generation of H2O2 from the catalyzing of GOD to glucose. In Ru(bpy)32+-TPrA system, the formation of H2O2 in situ on the electrode surface or the radical produced during the oxidation of H2O2 could annihilate the coreactant radical (TPrA˙) and thus the ECL signal significantly decreased. All the above results from EIS spectra and ECL responses both confirmed the successful fabrication of the sensing surface. In addition, the ECL signals were further applied to investigate the programmable DNA amplification ability based on the strand displacement reaction. As shown in Figure S2, ECL intensity in the presence of help DNA was quenched greatly compared with that of in the absence of help DNA, indicating that the detection signal could be amplified by the strand displacement reaction.

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Figure 3. (A) EIS spectra of the GCE at different stages. (a) bare GCE; (b) ABA/GCE; (c) En/ABA/GCE; (d) {RuSi NPs}4/En/ABA/GCE; (e) TN/{RuSi NPs}4/En/ABA/GCE; (f) BSA/TN/{RuSi NPs}4/ABA/GCE; (g) GOD-S/BSA/TN/{RuSi NPs}4/ABA/GCE. EIS were carried out in a background solution of 0.1 M KCl solution containing 5.0 mM Fe(CN)63-/Fe(CN)64- as electroactive probes. (B) Cyclic ECL intensity on potential curves of (a) {RuSi

NPs}4/En/ABA/GCE;

(b)

TN/{RuSi

NPs}4/En/ABA/GCE;

(c)

BSA/TN/{RuSi

NPs}4/En/ABA/GCE; (d) GOD-S/BSA/TN/{RuSi NPs}4/ABA/GCE. The modified electrodes were all measured in pH 7.4 PBS containing 30 mM TPrA and 10 mM glucose.

Analytical performance. In order to evaluate the sensitive and potential application of the proposed programmable DNA cyclic cascade amplifying ECL sensor coupled with DNA tetrahedral-based platform, various concentrations of target DNA were assayed. Under the optimized experimental conditions, the ECL signals of Ru(bpy)32+ decreased proportionally with the increasing concentrations of target DNA (Figure 4A). The ECL increment △I (△I = I0 – I, where I0 and I were ECL signals of BSA/TN/{RuSi NPs}4/ABA/GCE before (I0) and after (I) hybridization with GOD-S, respectively) was found to be logarithmically related to the concentration of target DNA in the range of 100 aM to 10 pM with a correlation coefficient of 0.994 (Figure 4B). The linear regression equation was y=1773.5x+28120.0 (y was the ECL increment △I and x was the logarithmical concentration of target DNA). The detection limit corresponding to a signal-to-noise ratio of 3 was 40 aM, which was superior to some existing DNA detection methods,36-38 implying the high sensitivity of the developed method. In addition, compared with some existing DNA detection methods, this proposed programmable DNA cyclic 17



cascade amplifying ECL sensor provides a more effective approach as well as wider dynamic concentration response range for the DNA detection (Table S2). The stability of this ECL sensor was examined by monitoring the ECL responses in Figure S3, which did not show obvious changes in ECL intensity. Besides, the reproducibility of ECL assay was evaluated from the response of a pretreated target DNA concentration of 10 pM for eight electrodes. A relative standard deviation (RSD) of ca. 6.5 % was obtained, indicating acceptable precision and high reproducibility of the develop methods. The selectivity of programmable DNA cascade amplifying ECL sensor was studied by measuring the ECL responses to three types of DNA sequences (target DNA, single-base mismatched DNA and three-base mismatched DNA). As shown in Figure 4C, in the presence of complementary target DNA (bar a), the integrated ECL (△I) was much bigger than that of single-base (bar b) or three-base (bar c) mismatched sequences. These results indicated that this developed programmable DNA cascade amplifying ECL sensing system exhibited an acceptable sequence specificity. Moreover, to evaluate the practical application of this sensing strategy, target DNA was spiked into 10 % human serum to simulate the situation of real sample detection and recovery testing was performed. For the spiked samples with 100 fM and 10 pM target DNA, the recoveries were 95% and 106%, respectively, demonstrating that the proposed ECL sensor had potential applications for DNA detection in real sample analysis.

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Figure 4. (A) ECL-time responses for the detection of various concentrations of target DNA (from a to g): 0, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, respectively. (B) The linear relationship between the relative ECL intensity (△I) and the logarithmic of target DNA concentration. ECL measurements were carried out in pH 7.4 PBS containing 30 mM TPrA and 10 mM glucose. Five measurements were applied for each point. (C) Bar graph of the integrated ECL intensity (△I = I0 - I) of BSA/TN/{RuSi NPs}4/ABA/GCE after being incubated with different DNA sequences (10 pM) and GOD-S. The different DNA sequences: (a) complementary sequence (target DNA), (b) single-base mismatched sequence and (c) three-base mismatched sequence, respectively.

■ CONCLUSION

In summary, we have developed a sensitive DNA tetrahedron-structured ECL biosensor for DNA assay via programmable DNA cyclic amplification. This present assay showed attractive features of (1) good selectivity. During programmable DNA amplification process, without free-energy release by new base-pair formation, the base pairs remained unchanged, thus avoiding the interference of other nucleic acid and showing outstanding recognition; (2) high sensitivity for target detection. The high hybridization efficiency of nanostructured electrode surface, the 19



catalysis of biological enzyme and programmable DNA cyclic amplification were all propitious to the improvement of sensitivity. Moreover, good accuracy, precision and sensitivity were obtained when this biosensor was applied in complex matrixes such as human serum samples, indicating a broad prospect in practical applications.

■ ASSOCIATED CONTENT Supporting Information DNA sequences employed in this work; Characterization of the DNA tetrahedron; Comparison of ECL signal with and without help DNA; The stability of ECL sensor; Comparison for existing DNA detection methods. The Supporting Information is available free of charge via the Internet at http:// pubs.acs.org.

■ AUTHOR INFORMATION Corresponding Author *Phone/Fax: +86-25-89687294. E-mail: [email protected]. Author Contributions All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

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■ ACKNOWLEDGMENTS

We appreciate the financial support of this work from Ministry of Science and Technology of China (No. 2016YFA0201200), the National Natural Science Foundation of China (Grant No. 21327902 and 21535003) and the Natural Science Foundation of Jiangsu Normal University (16XLR012).

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Self-Assembled DNA Tetrahedral Scaffolds for the Construction of

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