Sensitive Electrochemiluminescence Immunosensor for Detection of N

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Sensitive Electrochemiluminescence Immunosensor for Detection of N-acetyl-#-D-glucosaminidase Based on Novel “Light-Switch” Molecule Combined with DNA Dendrimer Haijun Wang, Yali Yuan, Ying Zhuo, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00357 • Publication Date (Web): 17 May 2016 Downloaded from http://pubs.acs.org on May 18, 2016

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Analytical Chemistry

Sensitive Electrochemiluminescence Immunosensor for Detection of N-acetyl-β-D-glucosaminidase Based on Novel “Light-Switch” Molecule Combined with DNA Dendrimer Haijun Wang, Yali Yuan, Ying Zhuo, Yaqin Chai∗, Ruo Yuan∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China * Corresponding author: Tel.: +86-23-68252277; Fax: +86-23-68253172. E-mail address: [email protected], [email protected]

1

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ABSTRACT Here, a novel “light-switch” molecule of Ru (II) complex ([Ru(dcbpy)2dppz]2+-DPEA) with self-enhanced electrochemiluminescence (ECL) property is proposed, which is almost nonemissive in aqueous solution, but is brightly luminescent when it intercalates into DNA duplex. Owing to less energy loss and shorter electron-transfer distance, the intramolecular ECL reaction between the luminescent [Ru(dcbpy)2dppz]2+ and coreactive tertiary amine group in N,N-Diisopropylethylenediamine (DPEA) makes the obtained “light-switch” molecule possess much higher light switched efficiency compared with traditional “light-switch” molecule. For increasing the loading amount and further enhancing the luminous efficiency of the “light-switch” molecule, biotin labelled DNA dendrimer (the fourth generation, G4) is prepared from Y-shape DNA by a step-by-step assembly strategy, which provides abundant intercalated sites for [Ru(dcbpy)2dppz]2+-DPEA. Meanwhile, the obtained nanocomposite (G4-[Ru(dcbpy)2dppz]2+-DPEA) could well bind with streptavidin labelled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreaction, an ECL immunosensor was fabricated for sensitive determination of N-acetyl-β-D-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The detemination linear range was 0.1 pg mL-1 ~ 1 ng mL-1 and the detection limit was 0.028 pg mL-1. The developed strategy combined the ECL self-enhanced “light-switch” molecular and DNA nanotechnology offers an effective signal amplification mean and provides ample potential for further bioanalysis and clinical study. KEYWORDS Electrochemiluminescence;

light-switch;

DNA

dendrimer;

2


Immunosensor;

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N-acetyl-β-D-glucosaminidase INTRODUCTION [Ru(bpy)2dppz]2+ and its analogues have aroused intense interest as a luminescent probe of DNA due to their extended aromatic structure which could intercalate into DNA duplex with high affinity.1,2 As classic “light-switch” molecule, this kind of complex is almost nonemissive in aqueous solution, but is brightly luminescent when it intercalates into DNA duplex.3 The generation of the “light-switch” effect mainly originates from the formation of hydrogen bond in aqueous solution, which would quench the luminescent excited-state and further reduce the quantum yield.4 However, after intercalation into DNA duplex, the phenazine nitrogens of those complexs were protected from the solvent, leading to an effective luminescent excited state. The obvious signal conversion would make this kind of complex extremely meaningful in wide application, for instance the study of other nonpolar microenvironments, the exploration of DNA and the design of sensors with low background. Considering their inherent excellent characteristics, it is very meaningful to further improve the light switched efficiency of [Ru(bpy)2dppz]2+ and its analogues by introducing some auxiliary functional approaches. Recently,

self-enhanced

electrochemiluminescence

(ECL)

reagent

containing

luminophore and coreactive group in the same molecular has received wide attention owing to its excellent luminous properties.5-8 Comparing to the traditional intermolecular reaction between luminophore and coreactive group, the intramolecular reaction of the self-enhanced ECL has obvious merits including shortened electronic transmission distance, reduced energy loss, improved luminous efficiency, decreased reagent consumption and measurement error.9

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Based on these consideration, a novel “light-switch” molecule with ECL self-enhanced property is proposed, which combined the improved luminescent efficiency of self-enhanced ECL with the excellent signal conversion property of “light-switch” molecule. It could be predictable that the novel “light-switch” molecule with ECL self-enhanced property will have excellent application potential in the construction of biosensors with low background and high sensitivity. Moreover, the light switched efficiency could be also enhanced by providing much more DNA duplex, thus increasing the intercalated amount of “light-switch” molecule. Currently, DNA nanostructures have attracted much attention, which could provide large amount of DNA duplex through molecular self-assemble nanotechnology.10,11 Until now, many well defined DNA nanostructures have been successfully assembled and extensively explored, such as ribbons,12 tetrahedrons,13 tubes,14 two- and three-dimensional extended crystals15 and so on. Among them, DNA dendrimers have received increasing concern due to their highly branched, globular, and nanosized structures with good monodispersity and outstanding stability.16,17 Simultaneously, they have great potential in acting as excellent nanocarriers for the construction of biosensors due to the easy operation, high biocompatibility, and sufficient stability. However, to date, it has been rarely reported that using DNA dendrimers as effective scaffolds to construct ECL biosensor. Herein, biotin labelled DNA dendrimer (the fourth generation, G4) was prepared from Y-shape DNA by a step-by-step assembly strategy. The large amount of DNA duplex in G4 provided

sufficient

intercalated

sites

for

Bis(4,4′-Dicarboxyl-2,2′-bipyridyl)

(4,5,9,14-Tetraaza-benzo[b]triphenylene) ruthenium(II) ([Ru(dcbpy)2dppz]2+), a derivative of

4


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the typical “light-switch” molecular of [Ru(bpy)2dppz]2+, by which the luminous efficiency of [Ru(dcbpy)2dppz]2+ was improved owing to the light switched property. Then, N,N-Diisopropylethylenediamine (DPEA), a typical tertiary amine as efficient coreactant to the ECL of Ru(II) complex, could be covalently crosslinked onto the obtained G4 composite through amidation reaction between carboxyl in [Ru(dcbpy)2dppz]2+ and amino group in DPEA. In this way, the luminous efficiency was further greatly improved by the formation of the ECL self-enhanced molecular ([Ru(dcbpy)2dppz]2+-DPEA). The obtained nanocomposite (G4-[Ru(dcbpy)2dppz]2+-DPEA) could well bind with streptavidin labelled detection antibody (SA-Ab2) due to the existence of abundant biotin. Through sandwiched immunoreactions, an ECL

immunosensor

was

prepared

for

sensitive

determination

of

N-acetyl-β-D-glucosaminidase (NAG), a typical biomarker for diabetic nephropathy (DN). The preparation of G4-[Ru(dcbpy)2dppz]2+-DPEA and the immunosensor were displayed in scheme 1. The development of the “light-switch” molecule with ECL self-enhanced property could expand the application of Ru(II) complex. Meanwhile, the strategy combined the highly specific recognition of immunoreactions with the effective signal amplification of DNA nanotechnology will also have a promising application in clinical detection.

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Scheme 1 A The preparation of G4-[Ru(dcbpy)2dppz]2+-DPEA, B Schematic diagram of the construction of the immunosensor and the response mechanism EXPERIMENTAL SECTION Reagents and apparatus. N,N-Diisopropylethylenediamine (DPEA) was purchased from

J&K

Scientific

Ltd.

(Beijing,

China).

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide

Chlorauric

hydrochloride

6


acid

(EDC),

(HAuCl4), N-hydroxy

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succinimide (NHS), streptavidin (SA) and bovine serum albumin (BSA) (96 ~ 99%) were got from Sigma-Aldrich (St. Louis, MO, USA). N-acetyl-β-D-glucosaminidase (NAG) and its antibody were purchased from HuaYi Bio-technology Co. Ltd. (Shanghai, China). Bis(4,4′-Dicarboxyl-2,2′-bipyridyl)(4,5,9,14-Tetraaza-benzo[b]triphenylene)

ruthenium(II)

([Ru(dcbpy)2dppz]2+) was acquired from Suna Tech Inc. (Suzhou, China). The serum speciments used in this work were got in southwest hospital (Chongqing, China). Tris-HCl buffer (20 mM, pH 8.3) was prepared with 20 mM Tris-HCl, 5 mM MgCl2 and 300 mM NaCl. Phosphate-buffered solution (PBS, pH 7.4, 0.1 M) was prepared with Na2HPO4 (0.1 M), KH2PO4 (0.1 M) and KCl (0.1 M). The oligonucleotides used in this work were prepared by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (Shanghai, China), and their sequences were shown in Table 1. Table 1. DNA sequences employed in our study Oligonucleotide

Sequences (from 5' to 3')

Y0a Y0b Y0c Y1a Y1b Y1c

GAC CGA TGG ATG ACC TGT CTG CCT AAT GTG CGT CGT AAG GAC CGA TGG ATG ACT TAC GAC GCA CAA GGA GAT CAT GAG GAC CGA TGG ATG ACT CAT GAT CTC CTT TAG GCA GAC AGG GAA GCC ACT CTG ACC TGT CTG ACT AAT GTG CGT CGT AAG GAA GCC ACT CTG ACT TAC GAC GCA CAA GGA GAT CAT GAG TCA TCC ATC GGT CCC CCC AGG TCT CAT GAT CTC CTT TAG TCA GAC AGG CTG TCA TCG GTC ACC TGT CTG CCT AAT GTG CGT CGT AAG CTG TCA TCG GTC ACT TAC GAC GCA CAA GGA GAT CAT GAG TCA GAG TGG CTT CCT CAT GAT CTC CT T TAG GCA GAC AGG GAC ACA CTG AGG TCC TGT CTG CCT AAT GTG CGT CGT AAG GAC ACA CTG AGG TCT TAC GAC GCA CAA GGA GAT CAT GAG TGA CCG ATG ACA GCT CAT GAT CTC CTT TAG GCA GAC AGG Biotin-TGC TGT CTG TCC ACC TGT CTG CCT AAT GTG CGT CGT AAG TGC TGT CTG TCC ACT TAC GAC GCA CAA GGA GAT CAT GAG ACC TCA GTG TGT CCT CAT GAT CTC CT T TAG GCA GAC AGG

Y2a Y2b Y2c Y3a Y3b Y3c Y4a Y4b Y4c

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For ECL monitoring, MPI-E ECL analyzer got from Xi’an Remax Electronic science & Technology Co.Ltd. (Xi’an, China) was applied. In determination, the potential scanning was 0 ~ 1.5 V, while the voltage of the photomultiplier tube (PTM) was 800 V. Three-electrode system (modified glassy carbon electrode (GCE), platinum wire, Ag/AgCl (sat. KCl)) was used in ECL detection. CHI 660E electrochemical workstation (Shanghai Chenhua Instrument, China) was used for electrochemical experiments. For characterization, transmission electron microscope (TEM, Hitachi Instrument, Japan) and atomic force microscopy (AFM, Bruker Co. USA) were used. Preparation of DNA Dendrimer. The dendrimeric DNA nanostructure was prepared by a self-assembled procedure according to the reference with some modification.16 Firstly, the Y-shaped DNA were prepared. For example, in the preparation of Y0, three oligonucleotide strands (Y0a, Y0b, Y0c) were mixed in Tris-HCl buffer (20 mM, pH 8.3, containing 5 mM MgCl2 and 300 mM NaCl) to reach a final concentration (5 µM) for each strand. For annealing, the obtained mixed solution was heated at 90 °C for 5 min and then slowly cooled to 4 °C. Subsequently, the annealed sample was incubated for another 30 min to form Y0 under room temperature. According to the similar procedure above, the other Y-shaped DNA (Y1, Y2, Y3, and Y4) were prepared. Afterward, the obtained Y-shaped DNA (Y0, Y1, Y2, Y3, and Y4) were directly used to assemble DNA dendrimers with different generations (Gn) that were prepared by mixing 1 molar of Gn-1 with 3 × 2n-1 molar of Yn. Generally, the first generation of DNA dendrimer (G1) was obtained by the hybridization of the sticky ends of Y0 (G0) and Y1 with a 1:3 molar stoichiometry for 1 h at room temperature, which leaving six free sticky ends available for further assembly. After that, the second generation of DNA

8


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dendrimer (G2) was formed through the further assembly between the as-prepared G1 and Y2 at a 1:6 molar stoichiometry. Through the iterative assembled process, DNA dendrimers with different generations could be prepared in few hours. As shown in scheme 1A, biotin labelled G4 was prepared to construct the immunosensor in our work. Preparation

of

G4-[Ru(dcbpy)2dppz]2+-DPEA.

The

preparation

of

G4-[Ru(dcbpy)2dppz]2+-DPEA was shown in scheme 1A. Firstly, 1 mL [Ru(dcbpy)2dppz]2+ (10 mM) was mixed with 2 mL of the obtained G4 solution. Owing to the extended aromatic structure, [Ru(dcbpy)2dppz]2+ could intercalate into DNA duplex of G4 with high affinity. Then, by assistance of EDC (0.2 M) and NHS (0.5 M), N,N-Diisopropylethylenediamine (DPEA, 97%, 500 µL), a typical tertiary amine as efficient coreactant to the ECL of Ru(II) complex, could be covalently crosslinked onto G4 through amidation reaction between carboxyl in [Ru(dcbpy)2dppz]2+ and amino group in DPEA, forming the composite of G4-[Ru(dcbpy)2dppz]2+-DPEA. In the process, the non-reacted reagents were removed by dialysis with centrifugal filter devices (8 KD). Construction of the ECL immunosensor. First of all, the GCE (Φ = 4 mm) was rinsed and sonicated with ethanol, deionized water respectively after polishing with 0.3 and 0.05 mm alumina slurry, continuously. Then, the GCE was immersed into HAuCl4 (1%) solution, then electrodeposited under -0.2 V for 30 s to form an Au nanoparticles (AuNPs) layer on the electrode. After that, the capture antibody (Ab1, 18 µL) was placed onto the electrode and incubated for 12 h at 4 °C. After rinsing, the electrode was placed into the solution of BSA for about 50 min for blocking the non-specific binding sites. For detection, the prepared immunosensor was immersed in NAG solution with different

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concentration for 45 min. Through sandwiched immunoreactions, streptavidin (SA) labeled detection antibody (Ab2, 200 µg mL-1) was modified on the electrode. After that, the prepared G4-[Ru(dcbpy)2dppz]2+-DPEA was captured through the specific binding between biotin and SA. Then, the modified electrode was put into the detection cell containing PBS (3 mL, 0.1 M, pH 7.4), and the ECL signals were recorded with MPI-E ECL analyzer. The ECL intensity was positively correlated with the concentration of NAG, by which NAG could be sensitively detected. The construction of the proposed immunosensor and the sensing of NAG were presented in scheme 1B. RESULTS AND DISCUSSION Morphology characterization of DNA Dendrimer. AFM and TEM were included for characterizing the morphology of the DNA Dendrimer (G4). As shown in Figure 1, both the AFM (A) and TEM (B) images illustrated that the G4 possessed a globular and branched structure with diameter of 60±5 nm.

Figure 1. AFM (A) and TEM (B) images of DNA dendrimer (G4) Possible ECL emitting mechanism of [Ru(dcbpy)2dppz]2+-DPEA. The possible luminous mechanism of [Ru(dcbpy)2dppz]2+-DPEA was deduced according to the previous 10


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reports.18 The possible mechanism was shown in Figure 2. Under potential scanning, both the luminescent [Ru(dcbpy)2dppz]2+ and the coreactive DPEA in [Ru(dcbpy)2dppz]2+-DPEA were oxidized

to

form

[Ru(dcbpy)2dppz]3+-DPEA•+.

Then,

the

intermediate

of

[Ru(dcbpy)2dppz]3+-DPEA• with strong activity was formed by the deprotonation. Through the

intramolecular

electron

transfer

and

energy

transmission,

the

obtained

[Ru(dcbpy)2dppz]3+-DPEA• turned to the excited state of [Ru(dcbpy)2dppz]*2+-DPEA. A strong ECL emission appeared when the excited state returned back to the ground state. Compared with the traditional intermolecular reaction, the ECL reaction occurred in intramolecular greatly shortened the electron-transfer path and reduced the energy loss, leading to a greatly amplified ECL signal.

Figure 2.The possible ECL mechanism of [Ru(dcbpy)2dppz]2+-DPEA ECL performance of the “light-switch” proceess. The light switched process was characterized by ECL. As shown in Figure 3, the modified electrode had a weak ECL response when [Ru(dcbpy)2dppz]2+ was put in aqueous solution due to the formation of

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hydrogen bond that would quench the excited-state (black curve). However, an obvious ECL signal was observed (about 22 times increase) when [Ru(dcbpy)2dppz]2+ intercalated into the prepared G4 (blue curve). After linking with DPEA through amidation reaction, [Ru(dcbpy)2dppz]2+-DPEA with self-enhanced ECL property was formed on the G4, resulting a further great enhancement of the ECL response (about 141 times increase, red curve). According to the experimental results, it can be confirmed that the proposed “light-switch” molecule with self-enhanced property possessed higher light switched efficiency, which would hold a prominsing application potential in clinical detection.

Figure 3. ECL profiles of the modified electrode with Ru(bpy)2(mcbpy)2+ in aqueous solution (a), [Ru(dcbpy)2dppz]2+ intercalated into DNA duplex of G4, (b) [Ru(dcbpy)2dppz]2+-DPEA intercalated into DNA duplex of G4 (c) Characterization of the stepwise fabrication. The electrochemical impedance spectroscopy (EIS) profiles of different modified electrodes were monitored in [Fe(CN)6]4−/3− (5 mM) containing KCl (0.1 M), which were displayed in Figure 4. An initial impedance value was acquired with the bare GCE (curve a), while it was decreased after deposition of AuNPs because of the promotion of AuNPs for electron transmission (curve b). Then, the

12


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impedance values were consecutively increased when Ab1 (curve c), BSA (curve d), NAG (curve e) and Ab2/G4-[Ru(dcbpy)2dppz]2+-DPEA (curve f) were incubated on the electrode due to the hindrance of protein for electron transmission.

Figure 4. EIS profiles of (a) bare GCE, (b) GCE/AuNPs, (c) GCE/AuNPs/Ab1, (d) GCE/ AuNPs/Ab1/BSA,

(e)

GCE/AuNPs/Ab1/BSA/NAG,

(f)

GCE/AuNPs/Ab1/BSA/NAG/

Ab2/G4-[Ru(dcbpy)2dppz]2+-DPEA in [Fe(CN)6]4−/3− (5 mM) containing KCl (0.1 M). Optimization of detected condition. The intercalated time of [Ru(dcbpy)2dppz]2+ was a capital factor for the performance of the proposed immunosensor. Thus, the optimal intercalated time of [Ru(dcbpy)2dppz]2+ was investigated. According to Figure 5, the ECL signal increased with extending the intercalated time until 5 h. Thus, we chose 5 h as the optimal intercalated time in this work.

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Figure 5. The ECL intensity diversified with the intercalated time of [Ru(dcbpy)2dppz]2+. All ECL signals were detected in PBS (pH 7.4, 0.1 M). The potential scan was 0 ~ 1.5 V and the PTM was 800 V. Detection of NAG with proposed immunosensor. From Figure 6, the ECL signal obviously increased with increasing the concentration of NAG (Figure 6A, curve a-i), and presented an excellent linear relationship with the logarithm of concentration (Figure 6B). The linear equation was I = 8453.6 + 1952.7 log c with a correlation coefficient of 0.9968, where I was ECL intensity and c was the concentration of NAG. The estimated limit of detection (LOD) was 0.028 pg mL-1, which was estimated by LOD = 3σb/k (σb was the standard deviation of the blank and k was the slope of the corresponding calibration curve19). Compared with the previous reports, the prepared immunosensor showed better performance as it was shown in Table 2, which illustrated that it would had a wider application in various bioassays.20-23

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Figure 6. A ECL profiles of the immunosensor incubating NAG with different concentrations of (a-i): 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5 and 1 ng mL−1. B Calibration plots of the proposed immunosensor. All ECL signals were detected in PBS (pH 7.4, 0.1 M). Table 2. Comparison of the present research with the previous works

Method

Target

Detection limit

References

Reflectometric interference spectroscopy

Amitriptyline

0.5 ng mL-1

20

Quartz crystal microbalance

Thrombomodulin

2 ng mL-1

21

Electrochemiluminescence

Antitransglutaminase type-2 antibodies

0.5 ng mL-1

22


NAG

0.17 pg mL-1

23


NAG

0.028 pg mL-1

This work

Performance of the proposed immunosensor. As several important performances, the reproducibility, stability and selectivity of the immunosensor were explored, respectively. At first, through consecutive cyclic potential scans for the immunosensor incubated with 0.005 ng mL-1 NAG, the stability of was explored. The relative standard deviation (RSD) of ECL peaks in 22 cycles was 1.9%, which suggested the good stability of the immunosensor (Figure

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7A). Then, the inter- and intra-assays were carried out to estimate the reproducibility. The inter-assay precision was explored with the ECL responses of three immunosensors incubated with 0.005 ng mL-1 NAG made at different batches. The intra-assay precision was investigated by detecting 0.005 ng mL-1 NAG with three proposed immunosensor made in the same batch. The calculated RSD were 3.52% and 4.43%, illustrating the outstanding reproducibility of the immunosensor (Figure 7B). Meanwhile, two interfering agents, hemoglobin (Hb) and Cyclin A2 (CA2), were used to study the selectivity. From Figure 7C, the ECL responses of the immunosensor incubating with pure Hb (1 ng mL-1) and CA2 (1 ng mL-1) were similar with that of the blank. Meanwhile, there were no obvious changes between the ECL responses got in the mixture (NAG (0.001 ng mL-1) and Hb (1 ng mL-1); NAG (0.001 ng mL-1) and CA2 (1 ng mL-1); NAG (0.001 ng mL-1), Hb (1 ng mL-1) and CA2 (1 ng mL-1)) and that obtained in pure NAG (0.001 ng mL-1). The comparison above illustrated the excellent selectivity of the immunosensor.

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Figure 7. A The ECL response of the immunosensor under consecutive cyclic potential scans; B Reproducibility (intra-assays and inter-assays) of the immunosensor. C Selectivity of the immunosensor for different targets: Blank; Hb (1 ng mL−1); CA2 (1 ng mL−1); NAG (0.001 ng mL−1); a mixture containing NAG (0.001 ng mL−1) and Hb (1 ng mL−1); a mixture containing NAG (0.001 ng mL−1), CA2 (1 ng mL−1); a mixture containing NAG (0.001 ng mL−1), Hb (1 ng mL−1) and CA2 (1 ng mL−1). Application of the immunosensor. Recovery experiment was performed by standard addition method in human serum to investigate the feasibility of the ECL immunosensor. Using the prepared immunosensor, NAG with different concentration added in human serum (defined as Added) was measured (defined as Found). The recovery was defined as the ratio between the Found concentration and the Added concentration, which would directly reflect the detected accuracy of the obtained immunosensor. From Table 3, the recoveries of the several detections are between 97.6% to 109%, demonstrating the good potential application of the immunosensor in clinical detection. Table 3. NAG Detection in human serum with the fabricated immunosensor

Sample number

Added/ng mL-1

Found/ng mL-1 (n = 3)

Recovery/%

1

0.500

0.488

97.6

2

0.100

0.103

103

3

0.050

0.0494

98.8

4

0.010

0.0109

109

CONCLUSIONS [Ru(dcbpy)2dppz]2+-DPEA, a novel “light-switch” molecule with self-enhanced ECL property was proposed in this work for the first time. And DNA dendrimer (the fourth 17



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generation, G4) prepared from Y-shape DNA by a step-by-step assembly strategy was used for the efficient immobilization of the “light-switch” molecule with large amount, leading to greatly improved ECL switched efficiency. Based on the obtained nanocomposite (G4-[Ru(dcbpy)2dppz]2+-DPEA), an ECL immunosensor was fabricated for sensitive detection of NAG, which displayed good detected performance in accuracy, selectivity, reproducibility, and stability. The construction of the immunosensor was significant for the monitoring of diabetic nephropathy, and the development of the “light-switch” molecule with ECL self-enhanced property and the strategy combined the highly specific recognition of immunoreactions with the effective signal amplification of DNA nanotechnology may hold promising potential in biological analysis and clinical applications. ASSOCIATED CONTENT Supporting Information Experimental details for XPS characterization for different complexs (Figure S1), Gel electrophoresis and DLS analysis for DNA dendrimers (Figure S2), and optimization of the concentration of [Ru(dcbpy)2dppz]2+ (Figure S3) AUTHOR INFORMATION * Corresponding authors Tel.: +86 23 68252277, fax: +86 23 68253172. E-mail addresses: [email protected], [email protected]. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT

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This work was financially supported by the NNSF of China (51473136, 21275119 and 21575116), Fundamental Research Funds for the Central Universities (XDJK2014C138). REFERENCES (1) Friedman A. E.; Chambron J. C.; Sauvage J. P.; Turro N. J.; Barton J. K. J. Am. Chem. Soc. 1990, 112, 4960-4962. (2) Stimpson S.; Jenkinson D. R.; Sadler A.; Latham M.; Wragg D. A.; Meijer A. J. H. M.; Thomas J. A. Angew. Chem. Int. Ed. 2015, 54, 3000-3003. (3) Brennaman M. K.; Alstrum-Acevedo J. H.; Fleming C. N.; Paul Jang, Meyer T. J.; Papanikolas J. M. J. Am. Chem. Soc. 2002, 124, 15094-15098. (4) Wachter E.; Howerton B. S.; Hall E. C.; Parkin S.; Glazer E. C.; Chem. Commun. 2014, 50, 311-313. (5) Swanick K. N.; Ladouceur S.; Zysman-Colman E.; Ding Z. F. Angew. Chem. Int. Ed. 2012, 51, 11079-11082. (6) Liang W. B.; Zhuo Y.; Xiong C.Y.; Zheng Y. N.; Chai Y. Q.; Yuan R. Anal. Chem. 2015, 87, 12363-12371. (7) Li P. P.; Jin Z. Y.; Zhao M. L.; Xu Y. X.; Guo Y.; Xiao D. Dalton Trans. 2015, 44, 2208-2216. (8) Chen A. Y.; Gui G. F.; Zhuo Y.; Chai Y. Q.; Xiang Y.; Yuan R. Anal. Chem. 2015, 87, 6328-6334. (9) Wang H. J.; He Y.; Chai Y. Q.; Yuan R. Nanoscale 2014, 6, 10316-10322. (10) Ko1 S. H.; Su M.; Zhang C.; Ribbe A. E.; Jiang W.; Mao C.D. Nat. Chem. 2010, 2, 1050-1055. 19



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

(11) Shapiro A.; Hozeh, A.; Girshevitz, O.; Abu-Horowitz, A .; Bachelet, I. Nucleic Acids Res. 2015, 43, 6587-6595. (12) Schulman R.; Winfree E. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15236-1524. (13) Li J.; Hong C. Y.; Wu S. X.; Liang H.; Wang L. P.; Huang G. M.; Chen X.; Yang H. H.; Shangguan D. H.; Tan W. H. J. Am. Chem. Soc. 2015, 137, 11210-11213. (14) Yin, P.; Hariadi, R.; Sahu, S.; Choi, H. M. T.; Park, S. H.; LaBean, T. H.; Reif, J. H. Science 2008, 321, 824-826. (15) Zheng, J. P.; Birktoft, J.; Chen, Y.; Wang, T.; Sha, R. J.; Constantinou, P.; Ginell, S.; Mao, C. D.; Seeman, N. C. Nature 2009, 461, 74-77. (16) Meng H. M.; Zhang X.B.; Lv Y. F.; Zhao Z. L.; Wang N. N.; Fu T.; Fan H. H.; Liang H.; Qiu L. P.; Zhu G. Z.; Tan W. H. ACS Nano 2014, 8, 6171-6181. (17) Zhou T.; Chen P.; Niu L.; Jin J.; Liang D. H.; Li Z. B.; Yang Z. Q.; Liu D. S. Angew. Chem. Int. Ed. 2012, 51, 11271-11274. (18) Zhuo Y.; Liao N.; Chai Y. Q.; Gui G. F.; Zhao M.; Han J.; Xiang Y.; Yuan R. Anal. Chem. 2014, 86, 1053-1060. (19) Radi A. E.; Sánchez J. L. A.; Baldrich E.; O’Sullivan C. K. J. Am. Chem. Soc. 2006, 128, 117-124. (20) Krieg A. K. and Gauglitz G. Anal. Chem. 2015, 87, 8845-8850. (21) Luo Y. Q.;, Liu T.; Zhu J. M.; Kong L. Y.;, Wang W.; Tan L. Anal. Chem. 2015, 87, 11277-11284. (22) Habtamu H. B.; Sentic M.; Silvestrini M.; Leo L. D.; Not T.; Arbault S.; Manojlovic D.; Sojic N.; Ugo P. Anal. Chem. 2015, 87, 12080-12087.

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(23) Wang H. J.; Yuan Y. L.; Zhuo Y.; Chai Y. Q.; Yuan R. Anal. Chem. 2016, DOI: 10.1021/acs.analchem.5b03954.

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For TOC only

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Sensitive Electrochemiluminescence Immunosensor for Detection of N

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