Intramolecular Self-Enhanced Nanochains Functionalized by an

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Intramolecular Self-Enhanced Nanochains Functionalized by ECL Reagent and Its Immunosensing Application for Detection of Urinary Beta2-Microglobulin Hui-Yun Yang, Haijun Wang, Cheng-Yi Xiong, Ya-Qin Chai, and Ruo Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b12011 • Publication Date (Web): 27 Sep 2017 Downloaded from http://pubs.acs.org on September 29, 2017

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ACS Applied Materials & Interfaces

Intramolecular Self-Enhanced Nanochains Functionalized by ECL Reagent and Its Immunosensing Application for Detection of Urinary Beta2-Microglobulin Hui-Yun Yang, Hai-Jun Wang, Cheng-Yi Xiong, Ya-Qin 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 ABSTRACT: In this study, polyethylenimine (PEI) is discovered to possess a noticeable amplification effect for the electrochemiluminescence (ECL) of N(aminobutyl)-N-(ethylisoluminol) (ABEI), and thus a novel self-enhanced ECL reagent (ABEI-PEI) is prepared by covalent crosslinking. Due to shortened electrontransfer path and reduced energy loss, the intramolecular ECL reaction between ABEI and PEI has exhibited enhanced luminous efficiency compared with traditional intermolecular ECL reaction. Owing to the amine-rich property of PEI, abundant ABEI could be immobilized on the molecular chains of PEI to strengthen the luminous intensity of ABEI-PEI. On account of the reducibility of remaining amino groups, ABEI-PEI, as the self-enhanced ECL reagent, has also been chosen as reductant and stabilizer for in-situ preparation of Au@Ag nanochains (Au@AgNCs) which has the catalytic activity for the ECL reaction. Moreover, taking ABEI-PEI as

∗

Corresponding authors at: Tel.: +86-23-68252277, fax: +86-23-68253172.

E-mail addresses: [email protected] (YQ. Chai); [email protected] (R. Yuan).

1

ACS Paragon Plus Environment


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template to directly prepare Au@AgNCs coinstantaneously achieves the immobilization of the ECL reagent with large amount. Meanwhile, in virtue of the electropositivity of ABEI-PEI capped Au@AgNCs (ABEI-PEI-Au@AgNCs), polyacrylic acid (PAA) with electronegativity is pervaded on the surface of nanochains and futher chelates with Co2+ to form ABEI-PEI-Au@AgNCsPAA/Co2+ complex, which could introduce Co2+ as catalyst to promote H2O2 decomposition, and thus oxidize ABEI to produce enhanced ECL signal. Here, the obtained self-enhanced ABEI-PEI-Au@AgNCs-PAA/Co2+ complex is utilized to capture the detection antibody (Ab2). According to sandwiched immunoreactions, a sensitive ECL immunosensor is constructed for the detection of beta2-microglobulin (β2-MG) with a wide linearity from 0.01 pg mL−1 to 200 ng mL−1 and a detection limit of 3.3 fg mL-1. KEYWORDS: Self-Enhanced ECL Reagent; in-situ Preparation; Functionalized Nanochains; Immunosensor; Urinary β2-MG 1.

Introduction

Chronic kidney disease (CKD), a cosmopolitan public health problem, has likely become one of the heaviest burden of public health resources, which could evolve into the mortal disease such as kidney failure and uremia without timely diagnose and treatment1, 2. Urinary beta2-microglobulin (β2-MG) is one biomarker of proximal tubular cell damage, and its concentration level presents positive correlation with the progression of CKD3. However, the daily output of β2-MG in human urine within the range of slight trace makes the sensitive and accurate detection an urgent requirement to validly assess the progression rate of CKD, 2


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considering that the result of its timely accurate detection could be regarded as a reliable detection index with regard to successful intervention treatment on nephrotic patients. Electrochemiluminescence (ECL), as a promising analytical method with wide applification in fundamental research4-6, has the superiority to serve the purpose of effectively detecting CKD, in view of its excellent properties such as high controllability, low background signal and high sensitivity7,8. Whereas, applying ECL in detection of β2-MG has been rarely reported yet. Among various ECL luminophores, for the intrinsic merits of chemical stability, nontoxicity, and high efficiency of luminescence, N-(aminobutyl)-N-(ethylisoluminol) (ABEI), a special analogue of luminol, has drawn intense attention in multiple fields9,10. Futhermore, compared with luminol, the spatial distance between the amino and the aromatic ring of ABEI is further, which is beneficial to avoid the conjugated attraction effect for amino, making it easier to be functionalized or immobilized and thus greatly narrowing the space length between luminescent label and electrode in order to improving the ECL efficiency11. ABEI-H2O2 system is defined as a classical ECL reaction system12,13. Using relevant catalysts can distinctly promote the decomposition of H2O2 and form reactive oxygen species (ROS) such as hydroxyl radical (OH•) and superoxide radical (O2•−), which can oxidize ABEI to produce enhanced ECL signal. Implementation of ECL signal amplification makes a key point in the biosensor fabrication14-16, which is conducive to enhancing the detection sensitivity of the biosensor. Wherein, self-enhanced ECL reagent, containing luminophore and coreactive group in one molecular via covalently linking has won wide attention owing to its excellent luminous efficiency17,18. In comparision to the traditional 3



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intermolecular ECL reaction between luminophores and coreactants, the intramolecular reaction in self-enhanced ECL system is more efficient owing to the shortened electron-transfer path and reduced energy loss, which is beneficial to significantly improve the ECL signal amplification. Polyethylenimine (PEI), an amine-rich polymer, has been applied broadly for its multiple intrinsic characteristics such as water solubility, anionic polyelectrolytes, complex formation with metal ions and so on19-21. In this report, an unique phenomenon was observed that PEI has an obvious enhancement effect for the ECL intensity of ABEI. Also of noted, according to reports in literatures, PEI can be utilized as reductant to in-situ form different PEI-conjugated nanomaterials, particularly in aspect of preparing PEI-conjugated Au, Ag nanoparticles (AuNPsPEIs, AgNPs-PEIs)22-24. Moreover, due to the cationic nature of PEI, NPs prepared by PEI are fully wrapped with cation which could drive electrostatic approaches to anionic entities (NPs-PEIs)25. Inspired by above multifarious characteristics of PEI, our work made an attempt of using PEI to immobilize abundant ABEI to form complex of ABEI-PEI with self-enhanced property, and then taking ABEI-PEI as template to synthesize a multifunctional nanomaterial with luminescent property. In this work, ABEI-PEI as a novel self-enhanced ECL reagent of high ECL efficiency is first prepared by crosslinking PEI with large amount of luminophore ABEI in assistance of glutaraldehyde (GA). Then, using the formed ABEI-PEI molecule as reductant and stabilizer, the Au@Ag nanochains (Au@AgNCs) are prepared

and

luminescence-functionalized,

which

simultaneously

realizes

immobilization of numerous ECL reagent. Subsequently, due to the electropositivity 4


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of Au@AgNCs reduced by amino groups, polyacrylic acid (PAA) with negatively charged carboxyl group (–COO-) is pervaded on the surface of the ABEI-PEIAu@AgNCs via electrostatic adsorption to make the nanochains more intensive. In addition, in virtue of the complexation reaction between Co2+ and –COO-, the remaining –COO- absorbing abundant Co2+ to form ABEI-PEI-Au@AgNCsPAA/Co2+ complex leads to a further ECL signal amplification, owing to the excellent catalysis of Co2+ to the ABEI-H2O2 system. Lastly, the obtained composites (ABEI-PEI-Au@AgNCs-PAA/Co2+) are applied as the immobilized platform of the detection antibody (Ab2) with bovine serum albumin (BSA) as the blocking agent of the nonspecific adsorption sites. In addition, Au nanoparticles (AuNPs) of prominent biocompatibility and electrocatalytic activity are applied to immobilize the capture antibody (Ab1). According to sandwiched immunoreactions, a sensitive ECL immunosensor is constructed to detect β2-MG. The preparation of PEI-Au@AgNCs-PAA/Co2+@Ab2 complex (Ab2 bioconjugates) and the fabrication procedure of the immunosensor were demonstrated in Scheme 1. The preparation of self-enhanced ECL reagent based nanochains would give an extension to the ECL application of ABEI in various areas. Simultaneously, the proposed immunosensor constructed favourably is expected to offer potential for disease diagnosis and clinical analysis.

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Scheme 1 Schematic Description of the Immunosensor Construction (A) and Response Mechanism (B). 2. Experimental 2.1 Preparation of the Ab2 bioconjugates (ABEI-PEI-Au@AgNCs-PAA/Co2+@Ab2BSA) The detailed preparation process of ABEI-PEI-Au@AgNCs-PAA/Co2+@Ab2BSA (Ab2 bioconjugates) was shown in Scheme 1A. First, 4 mL PEI (0.05%), 1.5 mL ABEI (0.005 M) were add into a 50 mL beaker and diluted in 5 mL double distilled water. And then, 100 µL GA (1%) was added and kept stirring for 2 h at room temperature (RT) to make PEI and ABEI cross-link sufficiently. Next, 50 µL 6


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fresh AgNO3 solution (0.25 M) was dropped into the ABEI-PEI solution with stirring and the reduction reaction was kept for 20 min at 50 °C until that the solution turned brownish yellow. As a result, AgNO3 was reduced by ABEI-PEI to form chain-like structure (ABEI-PEI-AgNCs). Afterward, 400 µL HAuCl4 (0.2 %) solution was added into the obtained ABEI-PEI-AgNCs solution. And then, the reaction system was kept stirring still about 2 h at 60 °C to get the nanochains denoted as ABEI-PEI-Au@AgNCs. The solution of the as-prepared ABEI-PEIAu@AgNCs was centrifuged at 12000 rpm for 15 min for discarding excess reagents. And the sediments were added into 2 mL double distilled water for dispersal. Subsequently, based on the electrostatic attraction between PEI and PAA, 200 µL PAA (0.05%) was added to form ABEI-PEI-Au@AgNCs-PAA. Then 20 µL CoCl2 (0.1 M) was added to form ABEI-PEI-Au@AgNCs-PAA/Co2+ nanoplatform, due to the complexation reaction between Co2+ and carboxyl (PAA). As-prepared ABEI-PEI-Au@AgNCs-PAA/Co2+ was centrifuged at 8000 rpm for 10 min and the sediments were dispersed in 2 mL double distilled water. After that, based on specific interaction between amino group and noble metal (Au@AgNCs), 200 µL anti-β2-MG solution (Ab2) was mixed into the obtained composites, and then reacted at 4 °C with stirring for 12h. In order to block the remaining active binding sites, 1 mL BSA (1%) was added into the obtained ABEI-PEI-Au@AgNCsPAA/Co2+@Ab2 complex with stirring. After centrifugation at 7000 rpm for 8 min to discard excess reagents, the bioconjugates of ABEI-PEI-Au@AgNCsPAA/Co2+@Ab2-BSA (Ab2 bioconjugates) was obtained. 2.2 Fabrication process of the modified electrodes

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Before modification, the GCE (4 mm in diameter) was polished with 0.3 and later with 0.05 µm alumina slurry. And then, the GCE was thoroughly cleaned with ethanol and double distilled water with 5 min ultrasonic processing. Next, the processed GCE immersed in HAuCl4 solution (1%) was electrochemical deposited (deposition potential: -0.2 V; deposition time: 30 s) to obtain a thin layer of Au nanoparticles (AuNPs). After drying, the electrode surface was incubated with 16 µL capture β2-MG antibody (Ab1) at 4 °C for 12 h. Then to block the nonspecific binding sites, 16 µL blocking agent BSA (1%) was placed onto the electrode surface for 40 min at RT. Subsequently, the modified electrode incubated with 16 µL β2MG antigen (Ag) was kept for 50 min with concentrations linearly increasing. Ultimately, the 16 µL Ab2 bioconjugates were modified on the obtained electrode through the immune binding between antigen and antibody. In procedure of the electrode fabrication, every step of the resultant electrode was softly washed by double distilled water to remove the excess and unreacted reagents. The Scheme 1 briefly described the fabrication of the modified electrodes and the reacted mechanism of the immunosensor. 3. Results and Discussion 3.1 Characterization of the nano-composite Firstly, the UV absorption spectroscopy was performed for proving the successful synthesis of ABEI-PEI-Au@AgNCs. According to Fig. 1, the UV-visible absorption spectra of PEI (a) was hardly found, while the absorption peaks of ABEI (b) was at 226, 283 and 322 nm. Compare to the absorption spectrum of PEI and ABEI, the UV-visible absorption spectra of ABEI-PEI (c) at 226 nm and 322 nm faded away 8


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and its absorption band around 290 nm became wider, owing to the crosslinking effect between ABEI and PEI. After using ABEI-PEI as reductant to prepare Au@AgNCs, there were absorption peaks (d) at 274 nm and 432 nm, which belonged to the ECL reagent (ABEI-PEI) and Au@Ag nanostructure, respectively, thus proving that the ECL reagent capped Au@AgNCs (ABEI-PEI-Au@AgNCs) was successfully prepared. Secondly, the prepared ABEI-PEI-Au@AgNCs and ABEI-PEI-Au@AgNCs-PAA were characterized by scanning electron microscope (SEM). According to Fig. 2A and Fig. 2a the Au@Ag nanochains obviously presented chain-like structures with an average diameter of 100 ± 20 nm, for the reason that reductant PEI was one kind of branched-chain polymer with linear structure and rendered the growth of nanoparticles along its lineament. As seen from Fig. 2B and Fig. 2b, owing to the electropositivity of Au@AgNCs and electronegativity of PAA, a more intensive status of the Au@Ag nanostructure was obtained through electrostatic adsorption between Au@AgNCs and PAA. Lastly, X-ray photoelectron spectroscopy (XPS) characterization was used to examine

the

integrated

preparation

of

ABEI-PEI-Au@AgNCs-PAA/Co2+

composites via elemental analysis. As shown in Fig. 3, the peaks at 532.48, 399.98, 284.48 eV belonged to O1s, N1s, C1s, respectively. Additionally, the doublets at 374.08, 367.88 eV and 87.88, 84.18 eV could assigned to Ag3d and Au4f, which proved the presence of Au@Ag nanochains. The peaks at 797.18, 781.28 eV belonged to Co2p. Further, high-resolution C1s and N1s XPS spectra have been performed to obtain more structural information of the proposed nano-composites. 9



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As seen from Fig.3 (h), raw data of C1s spectra contained two peaks of binding energy at 288.6 and 284.8 eV, which could be fitted by four components of C-C (284.7 eV), C-N (285.7 eV), C-O (286.8 eV), and O-C=O (288.6 eV), respectively, in high-resolution XPS spectra. In Fig.3 (i) of high-resolution N1s spectra, there were two peaks at 400.0 and 402.2 eV of raw data and could be fitted by two components of 399.9 and 402.1 eV, which were respectively associated with C-NH2 and -NR3+/-NH3+. Based on above-mentioned illustration, we could confirm the successful preparation of ABEI-PEI-Au@AgNCs-PAA/Co2+.

Fig. 1 UV-visible absorption spectrum of (a) PEI, (b) ABEI, (c) ABEI-PEI, (d) ABEI-PEI-Au@AgNCs.

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Fig. 2 SEM images of (A) ABEI-PEI-Au@AgNCs, (B) ABEI-PEI-Au@AgNCsPAA; SEM enlargement of (a) ABEI-PEI-Au@AgNCs, (b) ABEI-PEIAu@AgNCs-PAA.

Fig. 3 XPS analysis for (a) the full region of XPS for ABEI-PEI-Au@AgNCsPAA/Co2+ composites, (b) C1s region, (c) N1s region, (d) O1s region, (e) Ag3d 11



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region, (f) Au4f region, (g) Co2p region; High-resolution XPS spectra of C1s (h), N1s (i) for ABEI-PEI-Au@AgNCs-PAA/Co2+ composites. 3.2 Characterization of the ECL Immunosensor As shown in Fig. 4A, to indicate the successful fabrication of immunosensor stepwise, cyclic voltammetry (CV) was performed. The redox peak current of bare GCE was initially obtained (curve a), which was increased after modification of AuNPs layer by reason of the promotion effect of AuNPs for electronic transmission (curve b). Then, the redox peak currents were consecutively decreased when Ab1 (curve c), BSA (1%) (curve d), β2-MG (curve e) were incubated on the AuNPs/GCE surface due to the hindrance effect of protein layer for electronic transmission. To demonstrate the successful fabrication of Ab2 bioconjugate (ABEI-PEIAu@AgNCs-PAA/Co2+@Ab2-BSA), ECL behaviour was monitored in PBS (0.1 M, pH 8.0), which was displayed in Fig. 4B. According to Fig. 4B, ECL signal could be hardly detected before the modification of Ab2 bioconjugate (green curve). However, after incubating Ab2 bioconjugate (red curve), the ECL signal significantly increased due to the good luminous performance of the prepared ABEI-PEIAu@AgNCs-PAA/Co2+@Ab2-BSA probe.

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Fig. 4 (A) The CV characterization measured in [Fe(CN)6]3-/4- (pH 7.0, 5.0 mM) solution:

(a)

GCE,

(b)

AuNPs/GCE,

(c)

Ab1/AuNPs/GCE,

(d)

BSA/Ab1/AuNPs/GCE and (e) Ag/BSA/Ab1/AuNPs/GCE. (B) The ECL profiles of constructed immunosensor for 2 ng mL-1 β2-MG before (green) and after (red) the ABEI-PEI-Au@AgNCs-PAA/Co2+@Ab2-BSA bioconjugate modification in PBS (0.1 M, pH 8.0) at the PTM of 800 V and potential scanning of 0.2-0.8 V with the scan rate of 0.1 V s-1. 3.3 Comparison experiment By comparing ECL intensities of immunosensors with different Ab2 bioconjugates, contrast experiments were performed under the same condition for the purpose of demonstrating the advantage of the target probe. From Fig. 5, the change of the ECL intensity

(∆I)

of

the

immunosensor

with

(a)

ABEI-PEI-Au@AgNCs-

PAA/Co2+@Ab2-BSA (target bioconjugate) is 3412.5 a.u., while that decreased to 2907.8, 2574.9 and 1257.8 a.u. with immunosensor incubated with (b) ABEI-PEIAu@AgNCs@Ab2-BSA,

(c)

PEI-Au@AgNCs-ABEI@Ab2-BSA,

(d)

ABEI-

Au@AgNPs@Ab2-BSA, respectively. The reasons for the excellent performance of immunosensor with the target bioconjugate were stated as follows: (1) PEI could 13



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greatly enhance the ECL signal of ABEI due to its function of effective coreactant; (2) Compared with intermolecular coreaction, the intramolecular self-enhancement of ABEI-PEI reaction exhibited increased amplification for ECL signal; (3) The introduction of Co2+ had a good catalytic effect for H2O2 decomposition and further oxidized ABEI to produce enhanced ECL signal.

Fig. 5 ECL responses towards different Ab2 bioconjugates: (A) ABEI-PEIAu@AgNCs-PAA/Co2+@Ab2-BSA, (B) ABEI-PEI-Au@AgNCs@Ab2-BSA, (C) PEI-Au@AgNCs-ABEI@Ab2-BSA, (D) ABEI-Au@AgNPs@Ab2-BSA (2 ng mL−1 β2-MG for detection measured in PBS (0.1 M, pH 8.0) at the PTM of 800 V and potential scanning of 0.2-0.8 V with the scan rate of 0.1 V s-1). 3.4 Detection of β2-MG with the obtained immunosensor 14


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In terms of measuring β2-MG with the proposed immunosensor, it was clearly presented in Fig. 6 that the ECL signals gradually got increased along with the increasement of β2-MG concentration from 0.01 pg mL-1 to 200 ng mL-1 (Fig. 6A; curves a-h). The linear equation was I = 405.0 lg c + 3284.3 (I referred to ECL intensity; c referred to β2-MG concentration) and the square of correlation coefficient (R2) was 0.9956 with a detection limit estimated to be 3.3 fg mL-1 (Fig. 6B). Compared with previous works (Table 1), the proposed immunosensor showed relative lower detection limit, which illustrated that it might have a potential in highly sensitive bioassays.

Fig. 6 (A) ECL profiles of the immunosensor with different concentrations of β2MG (a-h) in PBS (0.1 M, pH 8.0): (a) 0.00001 ng mL-1, (b) 0.0002 ng mL-1, (c) 0.002 ng mL-1, (d) 0. 02 ng mL-1, (e) 0.1 ng mL-1, (f) 2 ng mL-1, (g) 20 ng mL-1 (h) 200 ng mL-1; (B) ECL calibration curve for β2-MG determination. 3.5 Related Performance The performance of the immunosensor was investigated through exploring its reproducibility, stability and selectivity in this study. From Fig. 7A, the 15



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reproducibility assay was investigated to value the relative standard deviations (R.S.D.). The R.S.D. of intra-assay and inter-assay revealed to be 3.46% and 4.53%, both below 5%, which demonstrated an excellent reproducibility of the proposed immunosensor. As seen from Fig. 7B, by incubating immunosensor with 2 ng mL-1 β2-MG for continuous scans for 11 cycles, the stability was explored to show a result of tiny change of ECL signal with R.S.D. of 3.22%, which suggested an excellent stability. Then, CEA and AFP were chosen as interference samples to investigate the selectivity of the immunosensor. Seen from Fig. 7C, the ECL responce of the immunosensor incubated with pure CEA has almost of no difference with that of blank sample. Furthermore, no obvious changes of ECL signal existed between the immunosensor incubated with pure β2-MG and the interfering sample (mixture 1, mixture 2), which indicated a superior selectivity for β2-MG detection.

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Fig. 7 (A) Reproducibility assays of four prepared immunosensor; (B) The ECL stability of prepared immunosensor under successive cyclic potential scans; (C) Specificity of immunosensor toward different targets: Blank; CEA (5 ng mL−1); β2MG (0.1 ng mL−1); a mixture containing β2-MG (0.1 ng mL−1) and CEA (5 ng mL−1); a mixture containing β2-MG (0.1 ng mL−1), CEA (5 ng mL−1), and AFP (5 ng mL−1). 3.6 Analysis of clinical urine samples By analyzing β2-MG in human urine samples from different people, the potential of this strategy for clinical application was explored. The whole urine specimens were collected from Southwest Hospital (Chongqing, China) and the pH of these urine specimens were adjusted within 6-8 in case of the degradation of β2-MG. The concentrations of β2-MG had been dertermined by hospital to obtain the reference value, and then the samples were stored at −20 °C for further detection. Before the analysis by this method, the samples of 200, 300, 500, 700 ng mL-1 were diluted 10 times by 0.1 M PBS (pH 7.4) to be 20, 30, 50, 70 ng mL-1 for determination. As seen from Table 2, the determination results of the two methods met an acceptable agreement, of which the relative error showed the range of -4.8–4.7%, which directly exhibited a favorable detected accuracy and an excellent potential in clinical detection. Table 1 Comparison of the Present Research with the Previous Works

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Table 2 Determination of β2-MG in Human Urine Samples

4. Conclusion In summary, luminescence-functionalized Au@Ag nanochains (ABEI-PEIAu@AgNCs-PAA/Co2+) with self-enhanced property were prepared for the ECL immunosensor construction to sensitively detect urinary β2-MG. This ECL sensing platform displayed several fascinating features: (1) The novel synthetical ECL reagent (ABEI-PEI) not only showed enhanced luminous efficiency, but also successfully immobilized abundant ABEI to strengthen the luminous intensity. (2) Au@AgNCs, with excellent catalytic activity to the ECL system, were in-situ prepared by using the obtained ECL reagent as reductant and stabilizer, which also realized the immobilization of massive ECL reagent. (3) ECL reagent capped 18


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Au@AgNCs (ABEI-PEI-Au@AgNCs) had the characteristic of electropositivity to adsorb PAA with electronegativity and futher chelate with Co2+, which exerted the catalysis of Co2+ to ECL system of ABEI-H2O2. In short, this method for preparing ECL reagent based nanostructures would broaden the application of luminescent in bioanalysis considering its multifunction, simplicity and effectiveness. ASSOCIATED CONTENT Supporting Information Materials and measurements; ECL performance of the immunosensor. ACKNOLEDGEMENT This work was financially supported by the NNSF of China (21575116, 21675129, 51473136) and the Fundamental Research Funds for the Central Universities (XDJK2017C023), and the China Postdoctoral Science Foundation (2016 M602626). REFERENCES [1] Drawz, P. E.; Rosenberg, M. E. Slowing Progression of Chronic Kidney Disease. Kidney Int. Suppl. 2013, 3, 372−376. [2] Jung, C. H.; Lee, M. J.; Kang, Y. M.; Hwang, J. Y.; Kim, E. H.; Park, J. Y.; Kim, H. K.; Lee, W. J. The Risk of Chronic Kidney Disease in a Metabolically Healthy Obese Population. Kidney Int. 2015, 88, 843−850. [3] Monteiro, M. B.; Thieme, K.; Santos-Bezerra, D. P.; Queiroz, M. S.; Woronik, V.; Passarelli, M.; Machado, U. F.; Giannella-Neto, D.; Oliveira-Souza, M.; CorrêaGiannella, M. L. Beta-2 microglobulin (β2M) Expression in the Urinary Sediment 19



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TOC Graphic


Intramolecular Self-Enhanced Nanochains Functionalized by an

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