Self-Enhanced Electrochemiluminescence ... - ACS Publications

Subscriber access provided by McMaster University Library

Article

Self-Enhanced Electrochemiluminescence Nanorods of Tris(bipyridine) Ruthenium (II) Derivative and Its Sensing Application for detection of N-acetyl-#-D-glucosaminidase Haijun Wang, Yali Yuan, Ying Zhuo, Yaqin Chai, and Ruo Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b03954 • Publication Date (Web): 18 Jan 2016 Downloaded from http://pubs.acs.org on January 19, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Analytical Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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

Analytical Chemistry

Self-Enhanced Electrochemiluminescence Nanorods of Tris(bipyridine) Ruthenium (II) Derivative and Its Sensing Application for detection of N-acetyl-β-D-glucosaminidase Haijun Wang, Yali Yuan, Ying Zhuo, Yaqin Chai∗, Ruo Yuan∗ Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, People’s Republic of China * Corresponding author: Tel.: +86-23-68252277; Fax: +86-23-68253172. E-mail address: [email protected], [email protected]

1

ACS Paragon Plus Environment


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

Page 2 of 27

ABSTRACT A self-enhanced electrochemiluminescence (ECL) reagent, synthesized by covalently linking

bis(2,2′-bipyridyl)(4′-methyl-[2,2′]bipyridinyl-4-carboxylicacid)

ruthenium(II)

(Ru(bpy)2(mcbpy)2+) with tris(3-aminopropyl)amine (TAPA), has been chosen as precursor to prepare nanorods ([Ru(bpy)2(mcbpy)2+-TAPA]NRs) with high luminous efficiency via a solvent evaporation induced self-assemble procedure. Due to the shorter electron-transfer path and less energy loss, the intramolecular reaction between the luminescent Ru(bpy)2(mcbpy)2+ and coreactive tertiary amine group in TAPA has shown improved luminous efficiency compared

with

the

common

intermolecular

ECL

reactions.

Moreover,

using

electrochemiluminescent Ru(II)-based complex as precursor to directly prepare nanostructure with high electro-active surface area, is a more effective and convenient method for enhancing the immobilized amount of Ru(II)-based complex in the construction of biosensors compared with the traditional immobilized ways. Meanwhile, the obtained nanorods could be further functionalized easily owing to their positive electrical property and amino-group on the surface. Here, Pt nanoparticles functionalized [Ru(bpy)2(mcbpy)2+-TAPA]NRs are used to load the detection antibody (Ab2). And the Au/Pd dendrimers (DRs) with hierarchically branched structures are synthesized to immobilize capture antibody (Ab1) with increased amount. Based on sandwiched immunoreactions, a simple and sensitive “signal-on” immunosensor is constructed for the detection of N-acetyl-β-D-glucosaminidase (NAG), a biomarker of diabetic nephropathy, with excellent linear in concentrations varying from 1 ng mL-1 to 0.5 pg mL-1 and a detection limit of 0.17 pg mL-1. KEYWORDS: Electrochemiluminescence; Self-enhanced nanorods; Au/Pd dendrimers;

2


Page 3 of 27

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


Immunosensor; N-acetyl-β-D-glucosaminidase INTRODUCTION Diabetic nephropathy (DN) is one of the most common chronic complications of diabetes mellitus (DM) that is likely to be the fifth leading cause of death worldwide, and much of the morbidity and mortality of diabetes can be attributed to nephropathy.1, 2 It has been proved that N-acetyl-β-D-glucosaminidase (NAG), which possesses direct correlation with the urinary excretion of transferrin, is an effective biomarker of DN.3 Therefore, the accurate and sensitive detection of NAG could assist in early assessment of the severity and progression rate of DN, which is meaningful for successful therapeutic intervention on diabetic patients. Electrochemiluminescence (ECL), as a powerful analysis tool generated by electrochemical reactions between electrogenerated species, could be effectively applied in NAG detection due to its excellent controllability, low background signal and simplified optical setup.4-8 Up to now, there almost has no relative report about this aspect. For enhancing the sensitivity of NAG determination with ECL, some assisted approaches and amplified strategies should be included. Immobilizing luminophore efficiently is a key point in the construction of ECL biosensors, which is directly associated with reaction amount and luminous efficiency of luminophore.9 In the early stage of ECL detection, the luminophores are directly put in the detection solution, which might waste the reagent and increase the cost, but get limited effectiveness. To overcome these drawbacks, the luminophores are usually immobilized on the electrode surface in the most of later researches, especially for the most commonly used ECL luminophores of tris(bipyridine) ruthenium (II) derivatives.10-12 For example, the

3



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

tris(bipyridine) ruthenium (II) derivative could be loaded on electrode surface in assistance of some organic solvents with excellent film forming property, for instance nafion, chitosan and N, N-dimethyllformamide.13, 14 In addition, tris(bipyridine) ruthenium (II) derivatives with different structures could also be immobilized on the electrode surface by using nanomaterials or DNA as supported platforms through adsorption, crosslinking, doping and embedding.15-16 In this way, the immobilized amount and luminous efficiency of tris(bipyridine) ruthenium (II) derivative are greatly enhanced, but still limited for the different immobilized efficiency. For further enhancing the luminous efficiency and avoiding tedious immobilization process, researchers tried to synthesize tris(bipyridine) ruthenium (II) derivative nanostructures with different shapes by directly using the tris(bipyridine) ruthenium (II) derivative as precursor, which would be applied as signal labels or immobilized platforms.17, 18 More attention about these tris(bipyridine) ruthenium (II) derivative nanostructures will be deserved owing to their high specific surface area, simple operation process, and high content of ruthenium luminophores. Self-enhanced ECL that makes luminophore and its coreactive group exist in the same molecular through covalently linking is a newly developed ECL reaction pattern with high luminous efficiency.19,

20

In this ECL reaction pattern, the electron transfer between

luminophore and the coreactive group occurs in intramolecular. Compared with the usually used intermolecular ECL reaction, it possesses the advantages in shortening the electronic transmission distance, improving the luminous stability, simplifying the operation, decreasing the measurement error and the using amount of reagent.21 More importantly, the intramolecular self-enhanced ECL reaction could obviously reduce the loss of energy caused

4


Page 4 of 27

Page 5 of 27

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


by the relaxation effect in the intermolecular reaction when the coreactants diffuse to the electrode surface, by which the luminous efficiency is enhanced significantly.22 Therefore, the self-enhanced ECL has been increasingly applied in the construction of biosensors for clinic detection with high sensitivity.23 In this work, in order to enhance the immobilized amount and luminous efficiency of luminescent agent simultaneously, we try to synthesize a self-enhanced tris(bipyridine) ruthenium (II) derivative nanostructure. Based on these descriptions above, it is conceivable that the self-enhanced ruthenium(II)-based nanostructure has more effective ECL reaction in accompany with extreme stable and strong ECL response. The preparation of the self-enhanced ruthenium(II)-based nanostructure with simple preparation process and excellent ECL property has significant meaning for expanding the ECL application in various field, especially for enhancing the detection sensitivity in sensing application. Herein, a ruthenium(II)-based self-enhanced molecule (Ru(bpy)2(mcbpy)2+-TAPA) is firstly

prepared

by

covalently

linking

the

luminophore

bis(2,2′-bipyridyl)(4′-methyl-[2,2′]bipyridinyl-4-carboxylicacid)ruthenium(II) (Ru(bpy)2(mcbpy)2+) with the coreactant tris(3-aminopropyl)amine (TAPA) together. Then, using the self-enhanced molecule as precursors, ruthenium(II)-based self-enhanced nanorods ([Ru(bpy)2(mcbpy)2+-TAPA]NRs) are

prepared

via a

solvent evaporation induced

self-assemble procedure. The obtained self-enhanced nanorods not only contain large amount of the self-enhanced luminophore, but also have high luminous efficiency due to intramolecular interaction between ruthenium(II)-based luminophore and coreactive tertiary amine groups. In addition, owing to positive electrical property and amino-group on the

5



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

Page 6 of 27

surface, Pt nanoparticles (PtNPs) with negative charge can be absorbed on the surface of the nanorods quite well to form [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs composite for improving the electrical conductivity. Then, the obtained composite could be used as the immobilized platform for the detection antibody (Ab2) through specific interaction between amino residues and PtNPs. Moreover, to increase the immobilized amount of capture antibody (Ab1), the Au/Pd dendrimers (DRs) with hierarchically branched structures are synthesized by sequential seed-directed overgrowth, which have high specific surface area and excellent electro-catalytic activity. Based on sandwiched immunoreactions, a sensitive “signal-on” ECL immunosensor

is

constructed

for

the

detection

of

NAG.

The

preparation

of

[(Ru(bpy)2(mcbpy)2+-TAPA)]NRs, Au/Pd DRs and the fabrication of the immunosensor were shown in scheme 1. The method for preparation of self-enhanced tris(bipyridine) ruthenium (II) derivative nanostructure proposed in this work would expand the application of ECL in various areas, and the construction of the immunosensor is significant for monitoring and therapeutics of diabetic nephropathy.

6


Page 7 of 27

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


Scheme 1 A The preparation of [(Ru(bpy)2(mcbpy)2+)-TAPA]NRs, B The fabrication of the immunosensor and the reacted mechanism EXPERIMENTAL SECTION Reagents and apparatus. N-acetyl-β-D-glucosaminidase (NAG) and its antibody were purchased

from

Shanghai

HuaYi

Bio-technology

Co.

Ltd.

(Shanghai,

China).

Ruthenium(II)-based complex was acquired from Suna Tech Inc. (Suzhou, China). Tris(3-aminopropyl)amine (TAPA) and trisodium citrate (Na3C6H5O7) were obtained from J&K Scientific Ltd. (Beijing, China).

L-Ascorbic

acid (L-AA), cetyltrimethylammonium

7



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

Page 8 of 27

bromide (CTAB), chlorauric acid (HAuCl4), palladium chlorine acid (H2PdCl4), chloroplatinic acid (H2PtCl6), Nafion (5%, V/V), 3-thiophenemalonic acid (TA), bovine serum albumin (BSA)

(96–99%),

N-hydroxy

succinimide

(NHS)

and

N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered solution (PBS) (pH 7.4, 0.1 M) was prepared with 0.1 M Na2HPO4, 0.1 M KH2PO4 and 0.1 M KCl. The serum speciments were got from Southwest Hospital (Chongqing, China). Deionized water was used throughout this study. Electrochemical experiments were performed on CHI 660E electrochemical workstation (Shanghai Chenhua Instrument, China). The ECL detection was carried out with MPI-E ECL analyzer (Xi’an Remax Electronic science & Technology Co.Ltd., Xi’an, China). In detection, the voltage of the photomultiplier tube (PTM) has been set at 800 V and the potential scanning is from 0 to 1.5 V. A three-electrode system with Ag/AgCl (sat. KCl) as reference electrode, platinum wire as counter electrode and the modified glassy carbon electrode (GCE) as working electrode, was used in the detection. For characterization, scanning electron microscopy (SEM, S-4800, Hitachi, Japan), X-ray photoelectron spectroscopy (XPS, Thermoelectricity Instruments，USA) were used. Preparation of Au/Pd DRs. The Au/Pd DRs with hierarchically branched structures were synthesized through sequential seed-directed overgrowth according to the literature with some modifications.24 Firstly, the octahedral Au seeds were prepared. CTAB (4.5 mL, 0.1M) was diluted in 24.6 mL deionized water in vial, following by adding HAuCl4 (500 µL, 0.01 M) and Na3C6H5O7 (500 µL, 0.1 M). The mixture solution was put in a 110 °C oil bath for six

8


Page 9 of 27

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


hours without stirring. The Au seeds were collected by centrifugation and re-dispersion. On the basis of Au seeds, the Au/Pd DRs were prepared through sequential seed-directed overgrowth. For nanocrystal growth, CTAB solution (2 mL, 0.2 M), H2PdCl4 solution (0.1 mL, 10 mM), HAuCl4 solution (206 µL, 0.1 M), L-AA solution (1.5 mL, 0.1 M), 21.4 mL deionized water and 1 mL prepared Au seeds were added serially. The reaction vial was gently shaken and introduced into a 25 °C oil bath for two hours without stirring. The Au/Pd DRs (G1) were obtained by centrifugation and re-dispersion. For successive generations, the process above was repeated using 1 mL the prepared G1 Au/Pd DRs as the seeds. The prepared process of Au/Pd DRs (G2) was shown in scheme 1 B. Preparation

of

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs.

The

preparation

of

[Ru(bpy)2(mcbpy)2+-TAPA]NRs was referenced the literature with some modifications.18 Firstly,

bis(2,2′-bipyridyl)(4′-methyl-[2,2′]bipyridinyl-4-carboxylicacid)

ruthenium

(II)

(Ru(bpy)2(mcbpy)2+) was obtained by refluxing cis-dichlorobis(2,2′-bipyridine) ruthenium(II) (Ru(bpy)22+) and 4′-methyl-[2,2′]bipyridinyl-4-carboxylicacid in an ethanol/water solution with protection of N2 for 12 h. With the assistance of EDC/NHS, the obtained Ru(bpy)2(mcbpy)2+ was covalently linked with TAPA, a classic tertiary amine, through amide bond to form Ru(bpy)2(mcbpy)2+-TAPA. Then, 50 mg Ru(bpy)2(mcbpy)2+-TAPA was dispersed in a mixed solvent of 15 mL acetonitrile and 5 mL n-propanol. Afterward, the mixed solution was heated at 100 °C, and the rod-like nanostructure was obtained by solvent evaporation

induced

self-assemble

procedure.

The

prepared

process

of

[Ru(bpy)2(mcbpy)2+-TAPA]NRs was shown in scheme 1 A. Finally, after centrifugation and re-dispersion, 2 mL [Ru(bpy)2(mcbpy)2+-TAPA]NRs further reacted with 500 µL Pt

9



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

Page 10 of 27

nanoparticles (PtNPs) to form [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs composite based on electrostatic adsorption and interaction between –NH2 and PtNPs. Fabrication of the ECL immunosensor. First of all, the bioconjugate of [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA was prepared. Concretely, 200 µL detection antibody (Ab2) was added in 2 mL [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs solution and

reacted

overnight

at

4

with

°C

stirring.

After

centrifugation,

the

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2 composite was obtained. To block the non-specific

adsorption

sites,

1

mL

BSA

was

mixed

with

1

mL

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2 with stirring. Followed by centrifugation at 9000

rpm

for

15

min

to

discard

excess

reagents,

the

bioconjugate

of

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA was obtained. Before modification, the GCE (Φ = 4 mm) was polished with 0.3, 0.05 mm alumina slurry continuously, and then rinsed thoroughly with deionized water and sonicated in ethanol, deionized water respectively. At first, 5 µL Au/Pd DRs (G2) dispersed with nafion (0.5%) was dropped onto the cleaned GCE. After drying, the electrode was incubated with 18 µL capture antibody (Ab1) for 12 h at 4 °C. Then, 18 µL BSA solution was placed on the electrode for 40 min to block the remaining active sites. After rinsing with deionized water to remove the excess and unreacted regent, the modified electrode was incubated in NAG solution for 45 min. Finally, the [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA bioconjugate was modified on the electrode through sandwiched immunoreaction. The fabrication and reacted mechanism of immunosensor was shown in scheme 1 B.

10


Page 11 of 27

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


RESULTS AND DISCUSSION Morphology characterization and elements analysis of different nanomaterials. The prepared [Ru(bpy)2(mcbpy)2+-TAPA]NRs and the Au/Pd DRs (G2) were characterized by SEM. According to Figure 1A, the [Ru(bpy)2(mcbpy)2+-TAPA]NRs obviously have rod-like structures with average length of 260 ± 30 nm and width of 100 ± 20. And the branched structures of Au/Pd DRs (G2) have also been observed form Figure 1B with a diameter of 150 ± 20 nm. Moreover, XPS characterization for elemental analysis was also performed for proving the successful preparation of different composites. As shown in Figure 1C, the peaks at 530.6, 398.6, 284.8 eV could be assigned to O1s, N1s, C1s respectively. And the doublets at 462.9 and 483.9 eV are belonged to Ru3p which proves the presence of Ru (II) luminophore. In the XPS spectra of [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs, the doublets of Pd4f (75.3 and 72.0 eV) appears obviously, suggesting the presence of PtNPs. From the XPS spectra analysis in Figure 1C, we could confirm the successful preparation of Ru(bpy)2(mcbpy)2+-TAPA and [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs.

Figure 1. A SEM image of [Ru(bpy)2(mcbpy)2+-TAPA]NRs, B SEM image of Au/Pd

11



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

dendrimers

(G2)

and

C

XPS

analysis

for

(a) the

Page 12 of 27

full

region

of

XPS

for

Ru(bpy)2(mcbpy)2+-TAPA and [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs, (b) Pt4f region, (c) Ru3p region, (d) O1s region, (e) C1s region, (f) N1s region Electro-active surface area of [Ru(bpy)2(mcbpy)2+-TAPA]NRs. The electro-active surface area will be enhanced when the ruthenium(II)-based complex self-assemble into rod-like structure, which plays an important role in improving the activity and reactivity of the electrode. To prove that, two GCE electrodeposited with gold nanoparticles (AuNPs) were modified with Ru(bpy)2(mcbpy)2+-TAPA and [Ru(bpy)2(mcbpy)2+-TAPA]NRs, respectively. Then, the cyclic voltammetry (CV) was recorded at different potential scan rates with [Fe(CN)6]4−/3− as redox probes. According to Figure 2, the redox peak currents have good linear relationship with the square root of scan rates in the range of 20-380 mV s−1. The regression equations are I = 638.1 v1/2 + 7.54 and I = 1017.22 v1/2 – 62.65, respectively. The electro-active surface areas of the two electrodes are 18.33 mm2 and 29.22 mm2, which are estimated according to the Randles-Sevcik equation:25 Ip = 2.69×105AD1/2n3/2v1/2c where Ip is the peak current, A is the apparent electrode area (cm2), D is the diffusion coefficient of the redox probe (D = 6.70 ± 0.02 × 10−6 cm2 s-1 at 25 °C), n is the number of electrons transferred in the reaction (n = 1), v is the scan rate of the CV measurement cycle voltammtric scanning rate (V s-1), and c is the concentration of the [Fe(CN)6]4−/3− (c = 5 mM). The difference of the electro-active surface areas demonstrates the excellent electrochemical activity of [(Ru(bpy)2(mcbpy)2+)-TAPA]NRs.

12


Page 13 of 27

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


Figure 2. A CVs and the linear relations of Ru(bpy)2(mcbpy)2+-TAPA and B [Ru(bpy)2(mcbpy)2+-TAPA]NRs modified GCE/AuNPs in 5.0 mM [Fe(CN)6]4-/3- at different scan rates from 20 to 380 mV s-1 Possible ECL emitting mechanism of [Ru(bpy)2(mcbpy)2+-TAPA]NRs. In the prepared

[Ru(bpy)2(mcbpy)2+-TAPA]NRs,

the

reaction

between

the

luminescent

Ru(bpy)2(mcbpy)2+ and coreactive tertiary amine group in TAPA occurs in intramolecular, which differentiates from the traditional intermolecular reaction. With the potential scanning from 0 to 1.5 V, both Ru(bpy)2(mcbpy)2+ and TAPA in [Ru(bpy)2(mcbpy)2+-TAPA]NRs are oxidized

to

[Ru(bpy)2(mcbpy)3+-TAPA•+]NRs.

form

And

the

[Ru(bpy)2(mcbpy)3+-TAPA•+]NRs loses proton to form [Ru(bpy)2(mcbpy)3+-TAPA•]NRs intermediate. With intramolecular electron transfer and energy transmission, the strong oxidant intermediate [Ru(bpy)2(mcbpy)3+-TAPA•]NRs changes to the excited state [Ru(bpy)2(mcbpy)*2+-TAPA]NRs which could further return back to the ground state with strong ECL emission producing. The possible ECL emitting mechanism is described as follows: [Ru(bpy)2(mcbpy)2+-TAPA]NRs  → [Ru(bpy)2(mcbpy)3+-TAPA•+]NRs (Oxidation) 13


(I)


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

Page 14 of 27

[Ru(bpy)2(mcbpy)3+-TAPA•+]NRs  → [Ru(bpy)2(mcbpy)3+-TAPA•]NRs + H+

(II)

[Ru(bpy)2(mcbpy)3+-TAPA•]NRs  → [Ru(bpy)2(mcbpy)*2+-TAPA]NRs (Self-coreaction)

(III)

[Ru(bpy)2(mcbpy)*2+-TAPA]NRs  → [Ru(bpy)2(mcbpy)2+-TAPA]NRs + hv (ECL emission) (IV)

ECL performance of [Ru(bpy)2(mcbpy)2+-TAPA]NRs. The ECL performance was characterized by ECL spectra. According to Figure 3A, the normalized ECL spectra of pure Ru(bpy)2(mcbpy)2+

has

a

maximum

wavelength

at

628

nm,

while

that

of

Ru(bpy)2(mcbpy)2+-TAPA is 638 nm. The shift of wavelength might be caused by the intramolecular

coreaction

between

Ru(bpy)2(mcbpy)2+

and

TAPA.

When

the

Ru(bpy)2(mcbpy)2+-TAPA accumulates to form rod-like structures, the maximum wavelength of the normalized ECL spectra is 644 nm, which is ascribed to the increased amount of self-enhanced luminescent agents and more effective energy transfer. To further clarify the difference of those different luminophores, the relationship between ECL response and potential was explored. As shown in Figure 3B, a peak of ECL response appears when the potential reaches 1.402 V in Ru(bpy)2(mcbpy)2+ solution (black curve). As comparison, the ECL peaks are acquired when the potentials reaches 1.365 V in Ru(bpy)2(mcbpy)2+-TAPA

solution

(red

curve)

and

1.337

V

in

[Ru(bpy)2(mcbpy)2+-TAPA]NRs solution (blue curve), respectively. The shifts of the ECL peak might be caused by the intramolecular reaction between luminophore and coreactive group in the obtained Ru(II)-based nanorods, which has faster electronic transfer and more

14


Page 15 of 27

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


effective energy transmission. Those comparisons results demonstrate that the self-enhanced nanorods have more effective ECL reaction.

Figure 3. A ECL spectra of Ru(bpy)2(mcbpy)2+ (green curve), Ru(bpy)2(mcbpy)2+-TAPA (red curve), [Ru(bpy)2(mcbpy)2+-TAPA]NRs (blue curve), respectively; B the relationship between the ECL responses and potentials in Ru(bpy)2(mcbpy)2+ (black curve), Ru(bpy)2(mcbpy)2+-TAPA (red curve) and [Ru(bpy)2(mcbpy)2+-TAPA]NRs (blue curve) solution, respectively Characterization of the ECL immunosensor. To demonstrate the successful stepwise fabrication of the immunosensor, electrochemical impedance spectroscopy (EIS) was performed in 5 mM [Fe(CN)6]4−/3− containing 0.1 M KCl, which was shown in Figure 4A. A small semicircle was observed on bare GCE (curve a), while that was increased after the modification of Au/Pd DRs dispersed with nafion because of electron hindrance of nafion (curve b). Subsequently, consecutive increase of the impedance value was obtained when the Ab1 (curve c), BSA (curve d) and NAG (curve e) were successively incubated on the electrode for the reason that the formation of protein molecules layers hindered the electron transfer.

15



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

Page 16 of 27

Moreover, the preparation of the immunosensor was characterized with ECL. As shown in

Figure

4B,

there almost no

ECL response before

the incubation

of the

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA bioconjugate because of the absence of luminophore (black curve). After the modification of the bioconjugate, however, the ECL signal greatly increased due to the introduction of luminophore and intramolecular coreaction amplification (red curve).

Figure 4. A EIS profiles of (a) Bare GCE, (b) GCE/Au/Pd dendrimers-nafion, (c) GCE/Au/Pd dendrimers-nafion/Ab1, (d) GCE/Au/Pd dendrimers-nafion/Ab1/BSA, (e) GCE/Au/Pd dendrimers-nafion/Ab1/BSA/NAG in 5 mM [Fe(CN)6]4−/3− containing 0.1 M KCl. B ECL profiles of the immunosensor in 0.5 ng mL−1 NAG before (black curve) and after (red curve) the modification of [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA bioconjugate Comparison of the immunosensor with different Ab2 bioconjugates. For comparing 16


Page 17 of 27

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


and demonstrating the advantages of the immunosensor with target Ab2 bioconjugate, four different nanocomposites were prepared to load Ab2 and BSA. The four resulted Ab2 bioconjugates bioconjugate),

were

(a) (b)

[Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA Ru(bpy)2(mcbpy)2+-TAPA-PtNPs@Ab2-BSA,

(target (c)

[Ru(bpy)2(mcbpy)2+-TAPA]NRs@Ab2-BSA, (d) [Ru(bpy)2(mcbpy)2+]NRs-PtNPs@ Ab2-BSA. The same batch of immunosensors were constructed and incubated with 0.5 ng mL−1 NAG, then coupled with the different Ab2 bioconjugates above, respectively. The changed ECL values (∆I) of the immunosensor with different Ab2 bioconjugates are shown in the Figure 5. The ∆I of the immunosensor incubated with target bioconjugate is 5491.8 (A), while that decreases to 3337.7, 2762.9 and 754.0 a.u. when the immunosensor is incubated with Ru(bpy)2(mcbpy)2+-TAPA-PtNPs@Ab2-BSA [Ru(bpy)2(mcbpy)2+-TAPA]NRs@Ab2-BSA

(B), (C),

[Ru(bpy)2(mcbpy)2+]NRs-PtNPs@Ab2-

BSA (D), respectively. The reasons for the excellent performance of immunosensor with the target bioconjugate are generalized as follows: (1) TAPA, as an effective coreactant for Ru(bpy)2(mcbpy)2+, could greatly amplify the ECL signal, especially through intramolecular coreaction; (2) The large loading amount of Ru(bpy)2(mcbpy)2+-TAPA in the nanostructures could induce a sharply enhanced ECL response; (3) PtNPs could also enhance the ECL signal by increasing the immobilization of Ab2 and promoting the electron transfer.

17



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

Figure 5. The ECL comparison between the immunosensor with different Ab2 bioconjugates: A [Ru(bpy)2(mcbpy)2+-TAPA]NRs-PtNPs@Ab2-BSA, B Ru(bpy)2(mcbpy)2+-TAPA-PtNPs @Ab2-BSA, C [Ru(bpy)2(mcbpy)2+-TAPA]NRs@Ab2-BSA, D [Ru(bpy)2(mcbpy)2+]NRs -PtNPs@Ab2-BSA (the immunosensor was incubated with 0.5 ng mL-1 NAG and measured in PBS (0.1 M, pH 7.4)) Optimization of analytical conditions. The incubation time between antibody and NAG could directly influence the performance of the immunosensor. To investigate the optimal incubation time, the ECL responses of the immunosensors incubated with 0.5 ng mL−1 NAG for different time were recorded. From Figure 6, the ECL intensity increases with increasing of the incubation time, and reaches a relatively stable value when the time is over than 45 min. Thus, 45 min was chose as the optimal incubation time in this work.

18


Page 18 of 27

Page 19 of 27

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


Figure 6. The relationship between ECL intensity and the incubation time of NAG Measurement of NAG with the proposed immunosensor. Under the optimized experimental conditions, the proposed immunosensor was used to detect NAG with a series of concentration. According to Figure 7A, the ECL intensity increases with increasing concentration of NAG from 0.5 pg mL-1 to 1 ng mL-1 (curve a-h), and has good positive correlation with the logarithm of concentration with a linear equation of I = 5959.4 + 1656.3 log c (where I is the ECL intensity and c is the concentration of NAG), and a squared correlation coefficient of 0.9979 (Figure 7B). The estimated limit of detection (LOD) (defined as LOD = 3SB/m, where SB is the standard deviation of the blank and m is the slope of the corresponding calibration curve26) was 0.17 pg mL-1. In addition, a performance comparison of the proposed biosensor with the previous reports was also carried out. From Table 1 we could see that the proposed immunosensor has relative lower detection limit, and might hold a new promise for highly sensitive bioassays applied in clinical detection.27-30

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

Page 20 of 27

Figure 7. A ECL profiles of the immunosensor in the presence of different concentrations of NAG (a-h): 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 1 ng mL−1. B ECL calibration curve of the immunosensor for NAG determination. All ECL signals were measured in PBS (0.1 M, pH 7.4). The PTM was set at 800 V and the potential scan was from 0 to 1.5 V. Table 1. Comparison of the present research with the previous works

Method

Target

Detection limit

References

Electrochemistry

Human apolipoprotein E4

0.3 ng mL-1

27

Electrochemistry

Epidermal growth factor receptor

50 pg mL-1

28

Reflectometric interference spectroscopy

Amitriptyline

0.5 ng mL-1

29

Electrochemiluminescence

Human IgG

0.05 ng mL-1

30

Electrochemiluminescence

NAG

0.17 pg mL-1

This work

The related performance of the immunosensor. To explore the selectivity of the immunosensor, hemoglobin (Hb) and prostate specific antigen (PSA) were chosen as interfering agents. According to Figure 8A, the ECL response of the immunosensor incubated

20


Page 21 of 27

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


pure Hb (10 ng mL-1) is almost the same as that of blank. And no obvious changes are observed compared the ECL intensity obtained in pure NAG (0.1 ng mL-1) with that obtained in the mixtures of NAG, Hb and PSA. Those results illustrate the superior selectivity of this immunosensor. Stability, another important performance of the immunosensor, was estimated under consecutive cyclic potential scans when the immunosensor was incubated with 0.5 ng mL-1 NAG. As shown in Figure 8B, the immunosensor has an excellent stability with relative standard deviations (RSD) of 2.2% of ECL peaks in 26 cycles. To further investigate the reproducibility, intra- and inter-assays were performed. From Figure 8C, the RSD (ECL response) of intra- and inter-assays are 3.66% and 4.69%, respectively, which demonstrates the excellent reproducibility of the proposed immunosensor.

Figure 8. A comparison of ECL responses with different targets: Blank; Hb (10 ng mL−1); NAG (0.1 ng mL−1); a mixture containing NAG (0.1 ng mL−1) and Hb (10 ng mL−1); a

21



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

Page 22 of 27

mixture containing NAG (0.1 ng mL−1), Hb (10 ng mL−1) and PSA (10 ng mL−1); B the ECL stability of proposed biosensor under consecutive cyclic potential scans; C The reproducibility of the proposed immunosensor Preliminary analysis of real samples. To explore the feasibility of clinical application, the recovery experiments were performed. NAG with different concentrations in the human serum were detected by the proposed immunosensor. The ratio between the actual concentration (defined as Added) and the detected concentration based on the linear equation (defined as Found) was the recovery. According to Table 2, the recoveries are 97.2% to 105.3%, which suggests that this obtained immunosensor could be effectively applied in clinical determination. Table 2. Determination of NAG in normal human serum with the proposed immunosensor

Sample number

Added/ng mL-1

Found/ng mL-1 (n = 3)

Recovery/%

1

1

1.053

105.3

2

0.5

0.492

98.4

3

0.1

0.0972

97.2

4

0.05

0.0511

102.2

CONCLUSIONS Ru(bpy)2(mcbpy)2+-TAPA, a self-enhanced ECL reagent with high luminous efficiency, was directly used to synthesize rod-like nanostructure through solvent evaporation induced self-assemble procedure, based on which an immunosensor was constructed for detection of NAG. By the formation of self-enhanced nanorods, the luminous efficiency and immobilized amount of Ru(bpy)2(mcbpy)2+-TAPA was greatly improved. With the aid of Au/Pd DRs, the constructed immunosensor could effectively detect NAG with excellent sensitivity, selectivity 22


Page 23 of 27

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


and reproducibility, holding a promise in the monitoring and therapeutics of diabetic nephropathy. Moreover, the method for preparing luminophore-based nanostructures would have a wider application in bioanalysis due to its simplicity, convenience and effectiveness. ASSOCIATED CONTENT Supporting Information Experimental details for Fourier transform infrared (FTIR) spectroscopy (Figure S1) and UV-vis absorption spectroscopy of different complexs (Figure S2) 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 This work was financially supported by the NNSF of China (51473136, 21275119 and 21575116), Fundamental Research Funds for the Central Universities (XDJK2014C138 and XDJK2015A002).

23



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

REFERENCES (1) Roglic G.; Unwin N.; Bennett P. H.; Mathers C.; Tuomilehto J.; Nag S.; Connolly V.; King H. Diabetes Care. 2005, 28, 2130-2135. (2) Peng R.; Liu H. D.; Peng H. M.; Zhou J.; Zha H.; Chen X.; Zhang L. Y.; Sun Y.; Yin P.; Wen L.; Wu T. H.; Zhang Z. Gene 2015, 570, 57-63. (3) Jha J. C.; Jandeleit-Dahm K. A.; Cooper M. E. Adv. Chronic Kidney Dis. 2014, 21, 318-326. (4) Liu Y. T.; Lei J. P.; Huang Y.; Ju H. X. Anal. Chem. 2014, 86, 8735-8741. (5) Hesari M.; Swanick K. N.; Lu, J. S.; Whyte R.; Wang S. N.; Ding Z. F. J. Am. Chem. Soc. 2015, 137, 11266-11269. (6) Wasalathanthri D. P.; Li D.; Song D. H.; Zheng Z. F.; Choudhary D.; Jansson I.; Lu X. L.; Schenkmand J. B.; Rusling J. F. Chem. Sci. 2015, 6, 2457-2468. (7) Xia H.; Li L. L.; Yin Z. Y.; Hou X. D.; Zhu J. J. ACS Appl. Mater. Interfaces 2015, 7, 696-703. (8) Ishimatsu R.; Matsunami S.; Kasahara T.; Mizuno J.; Edura T.; Adachi C.; Nakano K.; Imato T. Angew. Chem. Int. Ed. 2014, 53, 6993-6996. (9) Liu Z. Y.; Qi W. J.; Xu G. B. Chem. Soc. Rev. 2015, 44, 3117-3142. (10) Liu Z. Y.; Zhang W.; Qi W. J.; Gao W. Y.; Hanif S.; Saqib M.; Xu G. B. Chem. Commun. 2015, 51, 4256-4258. (11) Lin X. M.; Luo F. Q.; Zheng L.Y.; Gao G. M.; Chi Y. W. Anal. Chem. 2015, 87, 4864-4870. (12) Zhang J.; Qi H. L.; Li Y.; Yang J.; Gao Q.; Zhang C. X. Anal. Chem. 2008, 80,

24


Page 24 of 27

Page 25 of 27

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


2888-2894. (13) Wu M. S.; He L. J.; Xu J. J.; Chen H. Y. Anal. Chem. 2014, 86, 4559-4565. (14) Dang J.; Guo Z. H.; Zheng X. W. Anal. Chem. 2014, 86, 8943-8950. (15) Zhou C. S.; Luo J. J.; Zhang L.; Long Y. Y.; Wang C. M.; Xiao D. J. Mater. Chem. C 2014, 2, 9949-9956. (16) Xiong C. Y.; Wang H. J.; Liang W. B.; Yuan Y. L.; Yuan R.; Chai Y. Q. Chem. Eur. J. 2015, 21, 9825-9832. (17) Yu J. G.; Fan F. F.; Pan S. L.; Lynch V. M.; Omer K. M.; Bard A. J. J. Am. Chem. Soc. 2008, 130, 7196-7197. (18) Li Q.; Zheng J. Y.; Yan Y. L.; Zhao Y. S.; Yao J. N. Adv. Mater. 2012, 24, 4745-4749. (19) Swanick K. N.; Ladouceur S.; Zysman-Colman E.; Ding Z. F. Angew. Chem. Int. Ed. 2012, 51, 11079-11082. (20) Li P. P.; Jin Z. Y.; Zhao M. L.; Xu Y. X.; Guo Y.; Xiao D. Dalton Trans. 2015, 44, 2208-2216. (21) Chen A. Y.; Gui G. F.; Zhuo Y.; Chai Y. Q.; Xiang Y.; Yuan R. Anal. Chem. 2015, 87, 6328-6334. (22) Gui G. F.; Zhuo Y.; Chai Y. Q.; Xiang Y.; Yuan R. Anal. Chem. 2014, 86, 5873-5880. (23) Wang H. J.; He Y.; Chai Y. Q.; Yuan R. Nanoscale 2014, 6, 10316-10322. (24) Weiner R. G. and Skrabalak S. E. Angew. Chem. Int. Ed. 2015, 54, 1181-1184. (25) Li L. L.; Liu K. P.; Yang G. H.; Wang C. M.; Zhang J. R.; Zhu J. J. Adv. Funct. Mater. 2011, 21, 869-878. (26) Radi A. E.; Sánchez J. L. A.; Baldrich E.; O’Sullivan C. K. J. Am. Chem. Soc. 2006, 128,

25



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

117-124. (27) Liu Y. B.; Xu L. P.; Wang S. Q.; Yang W. Z.; Wen Y. Q.; Zhang X. J. Biosens. Bioelectron. 2015, 71, 396-400. (28) Ilkhani H.; Sarparast M.; Noori A.; Bathaie S. Z.; Mousavi M. F. Biosens. Bioelectron. 2015, 74, 491-497. (29) Krieg A. K. and Gauglitz G. Anal. Chem. 2015, 87, 8845-8850. (30) Han T. H.; Yan T.; Li Y. Y.; Cao W.; Pang X. H.; Huang Q. J.; Wei Q. Carbon 2015, 91 144-152.

26


Page 26 of 27

Page 27 of 27

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


For TOC only

27


Self-Enhanced Electrochemiluminescence ... - ACS Publications

Recommend Documents