Novel gold nanocluster electrochemiluminescence immunosensors based on nanoporous NiGd–Ni2O3–Gd2O3 alloys

Author’s Accepted Manuscript Novel gold nanocluster electrochemiluminescence immunosensors based on nanoporous NiGd-Ni2O3Gd2O3 alloys Xiaohui Lv, Hongmin Ma, Dan Wu, Tao Yan, Lei Ji, Yixin Liu, Xuehui Pang, Bin Du, Qin Wei www.elsevier.com/locate/bios

PII: DOI: Reference:

S0956-5663(15)30361-4 http://dx.doi.org/10.1016/j.bios.2015.08.038 BIOS7934

To appear in: Biosensors and Bioelectronic Received date: 15 June 2015 Revised date: 17 August 2015 Accepted date: 18 August 2015 Cite this article as: Xiaohui Lv, Hongmin Ma, Dan Wu, Tao Yan, Lei Ji, Yixin Liu, Xuehui Pang, Bin Du and Qin Wei, Novel gold nanocluster electrochemiluminescence immunosensors based on nanoporous NiGd-Ni2O3 Gd2O3 alloys, Biosensors and Bioelectronic, http://dx.doi.org/10.1016/j.bios.2015.08.038 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Novel gold nanocluster electrochemiluminescence immunosensors based on nanoporous NiGd-Ni2O3-Gd2O3 alloys Xiaohui Lv, Hongmin Ma, Dan Wu*, Tao Yan, Lei Ji, Yixin Liu, Xuehui Pang, Bin Du, Qin Wei

Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, PR China

∗Corresponding Author

Tel.: +86 0531 82767367 Fax: +86 531 82765969 E-mail: [email protected]

Abstract Herein, three-dimensional nanoporous NiGd alloy (NP-NiGd) was prepared by selectively dealloy Al from NiGdAl alloy in mild alkaline solution, then Ni2O3 and Gd2O3 grew further on the surface of NP-NiGd to obtain the NP-NiGd-Ni2O3-Gd2O3. On this basis, NP-NiGd-Ni2O3-Gd2O3 was further functionalized with gold nanoparticles (NP-NiGd-Ni2O3-Gd2O3@Au) and acted as sensor platform to fabricate a novel electrochemiluminescence (ECL) immunosensor. Bovine serum albumin protected gold nanoclusters (AuNCs@BSA) were prepared and acted as illuminant. AuNCs@BSA modified graphene oxide (GO/AuNCs@BSA) were used as labels of second antibody. In order to characterize the performance of the ECL immunosensor, carcino embryonie antigen (CEA) was used as the model to complete the experiments. Due to the good performances of NP-NiGd-Ni2O3-Gd2O3@Au (high surface area, excellent electron conductivity) and AuNCs@BSA (low toxicity, biocompatibility, easy preparation and good water solubility), the ECL immunosensor exhibited a wide range from 10-4 to 5 ng/mL with a detection limit of 0.03 pg/mL (S/N = 3). The immunosensor with excellent stability, acceptable repeatability and selectivity provided a promising method to detect CEA in human serum sample sensitively.

Key words: Gold nanocluster; Electrochemiluminescence; Immunosensor; CEA; NP-NiGd-Ni2O3-Gd2O3 alloy.

1. Introduction Organic or metallic organic compound luminescent materials, such as luminol, tris(2,2'-bipyridyl)ruthenium(II) chloride, have been applied in the fabrication of electrochemiluminescence (ECL) immunosensors (Zhao et al. 2015; Cao et al. 2012; Kim, Y., Kim, J. 2014; Chen et al. 2013). Quantum clusters are a new class of luminescent materials with only a few atoms in the core, attracting enormous attention due to their optical and electronic properties. In this work, bovine serum albumin (BSA) protected gold nanoclusters (AuNCs@BSA) were prepared and used as the luminescent material of ECL immunosensor. AuNCs@BSA exhibited molecule-like photophysical properties, large surface area-to-volume ratios and easy surface manipulation. AuNCs@BSA with the advantages of low toxicity, biocompatibility, easy preparation and good water solubility show great potential in theoretical studies and practical applications (Muhammed et al. 2010; Cui et al. 2014). Hollow metallic nanostructures represent a class of interesting materials with high surface area, low density and rich surface chemistry to allow function integration. Recently, dealloying strategy has been demonstrated to be very promising in scaling up the preparation of nanoporous metals (Xu et al. 2013; Biener et al. 2008; Xu et al. 2012). The interconnected skeleton extending in three dimensions of nanoporous metals can bypass the particle aggregation (Zhang et al. 2007; Xu et al. 2011). In addition, nanoporous metals prepared by dealloying have amounts of advantages such as excellent electron conductivity and extremely clean metal surface (Liu et al. 2011). Nanoporous PtCo alloy, nanoporous PtFe alloy, etc. have been successfully fabricated by dealloying ternary alloys (Xu et al. 2013; Li et al. 2014; Xu et al. 2013). It is well known that nanoporous binary metallic alloys combined the advantages of two metals have received considerable attention considering their synergistic catalytic and amplified effect (Wang et al. 2008). In this work, nanoporous NiGd-Ni2O3-Gd2O3 (NP-NiGd-Ni2O3-Gd2O3) alloy was

successfully fabricated by selectively etching the ternary NiGdAl source alloy in mild alkaline

solution.

The

gold

nanoparticles

(AuNPs)

functionalized

NP-NiGd-Ni2O3-Gd2O3 (NP-NiGd-Ni2O3-Gd2O3@Au) were applied as the platform of the ECL sensor. The nanocomposite of NP-NiGd-Ni2O3-Gd2O3@Au exhibited excellent electrochemical and catalytic activity for the luminescence of AuNCs@BSA. The performance of the proposed ECL immunosensor was characterized to estimate the amount of CEA. 2. Experimental Section 2.1. Materials and reagents HAuCl4

was

purchased

from

Shanghai

reagent

(Shanghai,

China).

Mercaptosuccinic acid (MSA) and bovine serum albumin (BSA, 96-99%) were purchased from Sigma-Aldrich (Beijing, China). All other chemicals were of analytical grade and used without further purification. The ultrapure water (≥18.25 MΩ) was used throughout the experiments. CEA and anti-CEA were purchased from Huaan Magnech Bio-Tech Co., Ltd. (Beijing, China). 2.2. Instruments The ECL emission measurements were carried out on a model MPI-F flow injection

chemiluminescence

detector

(Remax,

China)

and

electrochemical

measurements were carried out on CHI 760D electrochemistry workstation (Chenhua, China) at room temperature. Scanning electron microscope (SEM) images and energy dispersive spectrometer (EDS) were obtained using a field emission SEM (Zeiss, Germany). Electrochemical impedance spectroscopy (EIS) measurements were performed with IM6e Electrochemical Interface (Zahner, Germany). 2.3. Synthesis of NP-NiGd-Ni2O3-Gd2O3@Au alloys The NP-NiGd-Ni2O3-Gd2O3 alloys were synthesized according to Xu’s reported method (Xu et al. 2011) by dealloying the NiGd alloy foils in 2 M NaOH solution for 48 h at room temperature. Followed by rinsing thoroughly with doubly distilled water

and dried at room temperature in air. AuNPs were prepared by the method reported by Frens (Frens 1973). Briefly, 1 mL of HAuCl4 (1%) and 99 mL of ultrapure water were mixed adequately, then the solution was brought to reflux and 3 mL of sodium citrate solution (1%) was added dropwise under stirring. The reaction was then kept for another 15 min and cooled to room temperature. The claret-red AuNPs sol was obtained. NP-NiGd-Ni2O3-Gd2O3@Au alloys were synthesized by oscillating the solution (2 mL) containing 1 mL NP-NiGd-Ni2O3-Gd2O3 alloys (2 mg/mL) and 1 mL of AuNPs sol for 24 h. After AuNPs immobilized on the surface or the pores of NP-NiGd-Ni2O3-Gd2O3, the solution was centrifugated to remove the excess AuNPs. The obtained products were redispersed in 1 mL PBS (pH 7.4). 2.4. Preparation of AuNCs@BSA, GO/AuNCs@BSA Graphene oxide powders were synthesized from graphite according to the reported method (Marcano et al. 2010). AuNCs@BSA were prepared according to the process of reducing MSA capped AuNCs (MSA@AuNCs) with excess BSA (Muhammed et al. 2010). MSA@AuNCs were prepared according to the method reported by Yao’s group (Yao et al. 2001). As follows: 0.5 mmol of HAuCl4 (dissolved as aqueous solution in advance) and 0.5 mmol of MSA were mixed in 100 mL of methanol with bubbling of nitrogen gas at first, followed by the addition of a freshly prepared 25 mL of NaBH4 solution (0.2 mol/L) dropwise under vigorous stirring. The dark-brown precipitate was collected by centrifugation and thoroughly washed with water/ethanol (1:4) solution and ethanol to remove the inorganic and organic impurities. Finally, the obtained MSA@AuNCs were dried in vacuum. For the AuNCs@BSA synthesis, MSA@AuNCs and BSA (1:10 by weight) were mixed in water under stirring. 5 min later, the pH of the solution was adjusted to 12 with NaOH (2 mol/L) and stirring was allowed to continue for 6 h at 37 ºC. The AuNCs@BSA were purified by dialysis for 24 h with a water change after every 8 h and powder of AuNCs@BSA was collected by freeze-drying.

2.5. Preparation of NP-NiGd-Ni2O3-Gd2O3@Au/Ab1, GO/AuNCs@BSA and GO/AuNCs@BSA/Ab2 NP-NiGd-Ni2O3-Gd2O3@Au as substrate of the sandwich ECL immunosensor was used to load primary antibody (Ab1). NP-NiGd-Ni2O3-Gd2O3@Au/Ab1 was prepared by incubating the solution containing 2 mg of NP-NiGd-Ni2O3-Gd2O3@Au (dispersed in 1 mL of PBS, pH 7.4) and 0.01 mg of Ab1 under continuously oscillating at 4 ºC for 24 h. Ab1 can be loaded on NP-NiGd-Ni2O3-Gd2O3@Au via the chemical bond of Au and amino group (Mandal et al. 2005)). After that, NP-NiGd-Ni2O3-Gd2O3@Au/Ab1 was obtained by centrifugating and then redispersed in 1 mL of PBS (pH 7.4). GO/AuNCs@BSA acted as the labels of second antibodies (Ab2) were fabricated by oscillating the solution composed of 2 mg of GO and 2 mg of AuNCs@BSA for 48 h, followed by centrifugating and the obtained products were redispersed in 1 mL of PBS (pH 7.4). GO/AuNCs@BSA/Ab2 were fabricated by adding 0.1 mg of Ab2 into the above GO/AuNCs@BSA solution and incubating for 24 h at 4 ºC. After that, the solution was centrifugated and the products were redispersed in 1 mL of PBS (pH 7.4) and stored at 4 ºC for use. 2.6. Fabrication of the ECL immunosensor As shown in scheme 1, a glassy carbon electrode (GCE, 4 mm in diameter) was polished to a mirror with aqueous slurries of 1.0 μm, 0.3 μm and 0.05 μm α-Al2O3 powders on a polishing cloth. After rinsing with ultrapure water, 6 μL of NP-NiGd-Ni2O3-Gd2O3@Au/Ab1 (2 mg/mL) was dropped on the surface of the GCE and dried at 4

º

C, followed by modification 3 μL of BSA to the

NP-NiGd-Ni2O3-Gd2O3@Au/Ab1 film in order to block the nonspecific binding spots and dried at 4 ºC. After washing with buffer solution, the electrode was incubated with different concentration of CEA for 30 min at 4 ºC and then washed with buffer solution to remove the excess CEA. Finally, 6 μL of AuNCs@BSA/Ab2 was dropped on the electrode and incubated for 1 h. After washing, the ECL immunosensor was fabricated completely and was ready to be measured.

2.7. Measurement procedure The ECL detection was performed at room temperature in an ECL detector cell with 10 mL PBS (pH 7.4, 1/15 mol/L) containing 36 mM K2S2O8. A three-electrode system consisted of an Ag/AgCl (saturated KCl) electrode as the reference electrode, a platinum wire electrode as the auxiliary electrode and a modified GCE as the working electrode. A voltage of 800 V was supplied to the photomultiplier for luminescence intensity determination. In this study, the ECL intensity increased with the increasing concentrations of CEA, which may be ascribed to that more AuNCs@BSA were modified on the electrode acted as labels of Ab2. 3. Results and Discussion 3.1. Characterization of NP-NiGd-Ni2O3-Gd2O3@Au alloys, AuNCs@BSA, GO/AuNCs@BSA Figure

S1

shows

the

SEM

image

of

NP-NiGd-Ni2O3-Gd2O3

alloys.

Three-dimensional network nanostructure is the major characteristic which endows large surface area and electron transfer ability. The island-cracked structure can be deemed to an irregular nanoporous structure composed of pores with the size range of tens of nanometers and interconnected ligaments. The TEM image (Figure 1A) further indicates the formation of a three-dimensional network structure which is beneficial for the electron transport during ECL sensor. EDS was also used to infer the relative amounts of elements of NP-NiGd-Ni2O3-Gd2O3 by the relative peak heights of the EDS image (Figure 1B). The EDS image shows that Ni, Gd and O are contained in the nanoparticles, confirms that the nanoparticles were obtained successfully. To further verify the composition of the NP-NiGd-Ni2O3-Gd2O3, X-ray photoelectron spectroscopy (XPS) was employed to characterize the electronic structure of NP-NiGd-Ni2O3-Gd2O3. The XPS spectrum for NP-NiGd-Ni2O3-Gd2O3 shows complex structure 855.8 eV and 861.4 eV in the Ni2p region (Figure 2A), two strongest peaks at 141.4 eV and 146.2 eV in the Gd4d region (Figure 2B) and a single sharp peak at 529.5 eV in the O1s region (Figure 2C). The XPS spectra indicate the

existence of Ni2O3 and Gd2O3 in NP-NiGd-Ni2O3-Gd2O3 (Kim and Winograd 1974; Zhou et al. 2012; Cao et al. 2009). Figure 1C and Figure 1D show the SEM images of NP-NiGd-Ni2O3-Gd2O3@Au alloys. Obviously, large amounts of AuNPs were distributed on the surface and in the pores

of

NP-NiGd-Ni2O3-Gd2O3

uniformly,

which

indicated

that

the

NP-NiGd-Ni2O3-Gd2O3@Au alloys were obtained as expected. AuNCs@BSA and GO/AuNCs@BSA were also characterized by SEM. Clearly, AuNCs@BSA (Figure 1E) with uniform size and shape were synthesized as expectation. The EDS image (Figure S2A), UV-vis absorbance spectra (Figure S2B) and photoluminescence profiles (Figure S2C) can also support the formation of AuNCs@BSA. The SEM of GO/AuNCs@BSA (Figure 1F) proves that AuNCs@BSA distributed on the GO uniformly and GO/AuNCs@BSA nano-composites were prepared successfully. 3.2. Characterization of the modified electrode The proposed ECL modified electrode was characterized by the EIS. EIS is an effective method for monitoring the assembly of the immunosensor step by step and probing the feature of the surface modified electrode. The electron-transfer resistance (Ret) increased with increasing diameter of the semicircle (Wu et al. 2013). As shown in Figure 2D, bare GCE exhibited a very small Ret (curve a) due to the diffusional process. After the NPs-NiGd-Ni2O3-Gd2O3@AuNPs-Ab1 was modified on the electrode, though the Ab1 is protein which can hinder electron transfer, the Ret (curve b)

was

small

due

to

the

high

electrical

transport

properties

of

NPs-NiGd-Ni2O3-Gd2O3@AuNPs. Followed by blocking the nonspecific binding spots with BSA, the Ret (curve c) was increased which was attributed to that BSA was one kind of proteins which can hinder electron transfer between solution and electrode surface to a large extent. After Ab1 was allowed to incubate with CEA through immunoreactions, the modified electrode obtained a further increased Ret (curve d) due to the immune-complex film prevented the electron transfer to the electrode surface. The subsequent immobilization of GO/AuNCs@BSA/Ab2, the Ret (curve e)

was decreased which might be attribute to the excellent conductivity of GO/AuNCs@BSA. The results were consistent with the fact that the electrode was modified as expected. 3.3. Optimization of the conditions The performance of the ECL immunosensor is usually related to the kind of coreactant, pH of the base solution and the concentration of coreactant. In order to obtain the excellent ECL signal, they were investigated as follows. H2O2 and K2S2O8 are two common reagents usually act as coreactant. Herein, the ECL signals of AuNCs@BSA with H2O2 and K2S2O8 acted as coreactant respectively were compared by modifying GO/AuNCs@BSA on the GCE. Though the ECL signals of AuNCs@BSA with H2O2 (Figure 3A) acted as coreactant was stable, the background was great. While the ECL signals of AuNCs@BSA with K2S2O8 (Figure 3B) acted as coreactant was not only stable, but the background was ignored. Therefore, K2S2O8 was selected as the coreactant in the following experiment. The effect of pH value to the performance of the ECL immunosensor was studied. The ECL signals of the proposed ECL immunosensor in PBS at various pH values ranging from 6.0 to 9.0 are shown in Figure 3C. It can be seen that the ECL signals showed the trend from increase to decrease with the increasing pH values from 6.0 to 9.0. The ECL signal achieved to maximum at pH 7.4. This may attribute to that the luminescence was inhibited because of the proton could be reduced easily at negative potential at low pH. In addition, the strong oxidant SO4•− was consumed via scavenging effect of OH- at high pH (Yao et al. 2008; Xu et al. 2000). Therefore, PBS with pH 7.4 was used as the working solution. The effect of K2S2O8 concentration to the performance of the ECL immunosensor was also studied in the range from 20 to 45 mM. As shown in Figure 3D, in the condition of more excited states of AuNCs@BSA produced which resulted by the facilitation of S2O82-, the ECL signals increased in K2S2O8 concentration range from 20 to 36 mM and reached maximum at 36 mM. The ECL signals decreased when the concentration of K2S2O8 was more than

36 mM, which might be attributed to that more S2O82- could inhibit the luminescence of AuNCs@BSA. Therefore, 36 mM was selected as the optimized concentration of K2S2O8 in this work. 3.4. Performance of the proposed ECL modified electrode to CEA 3.4.1. Calibration curve Under the optimized conditions, the sensitivity and dynamic range of the developed ECL immunosensor were evaluated toward CEA in pH 7.4 PBS containing 36 mM of K2S2O8 with a sandwich-type ECL immunoassay format. As seen in Figure 4, the ECL intensity increased with the increase of CEA concentration. A linear dependence between the ECL signals and the logarithm of CEA concentration was obtained in the range from 10-4 to 5 ng/mL with a detection limit of 0.03 pg/mL (S/N = 3). The linear regression equation was I = 2407 – 356 logc with a correlation coefficient of R2 = 0.9934. Compared with the detection limit of amperometric immunosensor (0.06 ng/ml) (He et al. 2008), electrochemical immunosensor (3.5 pg/mL) (Lu et al. 2014), fluorescent

sensors

(5

pg/mL)

(Zhou

et

al.

2014)

and

capillary

electrophoresis-chemiluminescence (4.8 pg/mL) (Zhou et al. 2015), the proposed ECL immunosensor has a lower detection limit. The lower detection limit may be attributed

to

the

following

factors:

(1)

the

large

surface

area

of

NPs-NiGd-Ni2O3-Gd2O3 and GO/AuNCs@BSA could fix a large amount of AuNPs and

anti-CEA;

(2)

the

excellent

conductivity

and

catalytic

activity

of

NPs-NiGd-Ni2O3-Gd2O3@AuNPs and GO/AuNCs@BSA could greatly improve the ECL signal. 3.4.2. Stability and repeatability of the ECL immunosensor As shown in Figure S3A, the stability of the proposed ECL immunosensor was evaluated under consecutive cyclic potential scans for 13 cycles for the detection of CEA (0.1 ng/mL). There are no obvious changes in the ECL intensity and the relative standard deviation (RSD) was 2.38%. The excellent stability may be attributed to that

NPs-NiGd-Ni2O3-Gd2O3@AuNPs and GO/AuNCs@BSA provided good platforms for the loading of Ab1 and Ab2, which could increase the access chance of the antigen and antibody. The storage stability of the proposed immunosensor was also satisfactory (Figure S4). In order to investigate the repeatability of the sensor, five ECL immunosensors were prepared for detecting 0.1 ng/mL of CEA. The RSD of the ECL signals (Figure S3B) was 2.3%. 4. Application for CEA samples The possible applicability of the developed ECL immunosensor for real samples was investigated by detecting CEA of human serum using the standard addition method. The results were calculated according to the calibration curve mentioned above and the results are listed in Table 1. The recovery of the proposed sensor was in the range from 98.26% to 102.9% revealing a high method accuracy of the proposed ECL immunosensor and it is has the potential application in practical sample testing. 5. Conclusions In summary, a novel nanoporous NP-NiGd-Ni2O3-Gd2O3 alloy was prepared by a simple dealloying strategy for ECL immunosensor fabrication. The proposed metallic nanostructure with high surface area, excellent electron conductivity allowed a large amount of biomolecules were loaded and enhanced the ECL signal intensity of AuNCs@BSA. The ECL immunosensor can be used for the sensitive detection of CEA in a wide concentration range with low detection limit. The nanoporous thermometal alloys with hollow ligaments can retain the biocatalytic activity and stability of the metals, indicating that it has great application potential in ECL immunosensor. Acknowledgements This study was supported by the Natural Science Foundation of China (No. 21175057, 21375047, 21377046), the Science and Technology Plan Project of Jinan (No. 201307010), the Science and Technology Development Plan of Shandong

Province (No. 2014GSF120004), and Q. Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province and UJN (No. ts20130937).

Figure caption Scheme 1. The fabrication process of the ECL modified electrodes. Figure 1. TEM (A) and EDS (B) images of NP-NiGd-Ni2O3-Gd2O3; SEM images of NP-NiGd-Ni2O3-Gd2O3@Au (C and D); AuNCs@BSA (E) and GO/AuNCs@BSA (F). Figure 2. XPS of NP-NiGd-Ni2O3-Gd2O3 in the Ni2p (A), Gd4d (B) and O1s (C) regions; EIS of the proposed ECL immunosensor (D). Figure 3. The selection of coreactants between H2O2 (A) and K2S2O8 (B), the effects of pH values (C) and concentrations of K2S2O8 (D). Figure 4. Calibration curve for CEA determination. Medium: 1/15 M PBS (pH 7.4) containing 36 mM K2S2O8 under single-step cycle pulse.

Table caption Table 1. The recovery of CEA in the real samples using the proposed ECL immunosensor measured in 10 mL of PBS (pH 7.4) containing 36 mM K2S2O8.

Reference Biener, J., Nyce, G. W., Hodge, A. M., Biener, M. M., Hamza, A. V., Maier, S. A., 2008. Advanced Materials, 20(6), 1211-1217. Cao, X., Li, X., Gao, X., Yu, W., Liu, X., Zhang, Y., Cheng, X., 2009. Journal of Applied Physics, 106(7), 073723. Cao, Y., Yuan, R., Chai, Y., Mao, L., Niu, H., Liu, H., Zhuo, Y., 2012. Biosensors and Bioelectronics, 31(1), 305-309. Chen, Z., Liu, Y., Wang, Y., Zhao, X., Li, J., 2013. Analytical Chemistry, 85(9), 4431-4438. Cui, M., Zhao, Y., Song, Q., 2014. TrAC Trends in Analytical Chemistry, 57, 73-82. D.C. Marcano, D.V. Kosynkin, J.M. Berlin, A. Sinitskii, Z. Sun, A., 2010. ACS Nano, 4, 4806–4814. Frens, G., 1973. Nature, 241(105), 20-22. He, X., Yuan, R., Chai, Y., Shi, Y., 2008. Journal of biochemical and biophysical methods, 70(6), 823-829. Kim K S, Winograd N., 1974. Surface Science, 43(2): 625-643. Kim, Y., Kim, J., 2014. Analytical Chemistry, 86(3), 1654-1660. Liu, A., Geng, H., Xu, C., Qiu, H., 2011. Analytica Chimica Acta, 703(2), 172-178. Li, Y., Xu, C., Li, H., Wang, H., Wu, D., Ma, H., Wei, Q., 2014. Biosensors and Bioelectronics, 56, 295-299. Lu, W., Qian, C., Bi, L., Tao, L., Ge, J., Dong, J., Qian, W., 2014. Biosensors and Bioelectronics, 53, 346-354.

Mandal, S., Phadtare, S. Sastry, M., 2005. Current Applied Physics, 5, 118-127. Muhammed, M. H., Verma, P. K., Pal, S. K., Retnakumari, A., Koyakutty, M., Nair, S., Pradeep, T., 2010. Chemistry-A European Journal, 16, 10103-10112. Wang, J., Thomas, D. F., Chen, A., 2008. Analytical Chemistry, 80(4), 997-1004. Wu, B., Hu, C., Hu, X., Cao, H., Huang, C., Shen, H., Jia, N., 2013. Biosensors and Bioelectronics, 50, 300-304. Xu, C., Hou, J., Pang, X., Li, X., Zhu, M., Tang, B., 2012. International Journal of Hydrogen Energy, 37(14), 10489-10498. Xu, C., Liu, A., Qiu, H., Liu, Y., 2011. Electrochemistry Communications, 13(8), 766-769. Xu, C., Liu, Y., Hao, Q. Duan, H., 2013. Journal of Materials Chemistry A, 1(43): 13542-13548. Xu, C., Liu, Y., Su, F., Liu, A., Qiu, H., 2011. Biosensors and Bioelectronics, 27(1), 160-166. Xu, C., Sun, F., Gao, H., Wang, J., 2013. Analytica Chimica Acta, 780, 20-27. Xu, G., Dong, S., 2000. Electroanalysis, 12, 583-587. Yao, H., Momozawa, O., Hamatani, T., Kimura, K., 2001. Chemistry of Materials, 13(12), 4692-4697. Yao, W., Wang, L., Wang, H., Zhang, X., 2008. Electrochimica Acta, 54, 733-737. Zhang, J., Sasaki, K., Sutter, E., Adzic, R. R., 2007. Science, 315(5809), 220-222. Zhao, H. F., Liang, R. P., Wang, J. W., Qiu, J. D., 2015. Biosensors and Bioelectronics, 63, 458-464. Zhou, Z. M., Feng, Z., Zhou, J., Fang, B. Y., Qi, X. X., Ma, Z. Y., Hu, X. B., 2015. Biosensors and Bioelectronics, 64, 493-498.

Zhou, L., Gu, Z., Liu, X., Yin, W., Tian, G., Yan, L., Zhao, Y., 2012. Journal of Materials Chemistry, 22(3): 966-974. Zhou Z.M., Zhou J., Chen J., Yu R.N., Zhang M.Z., Song J.T., Zhao Y.D., 2014. Biosensors and Bioelectronics, 59, 397-403.

Highlights ● NP-NiGd-Ni2O3-Gd2O3 synthesized by dealloying method was firstly used to

fabricate ECL modified electrodes. ●

ECL

immunosensor

based

on

gold

nanoparticles

NP-NiGd-Ni2O3-Gd2O3 was fabricated. ● The nanocomposites showed high catalytic activity. ● It owned ultrahigh sensitivity, good stability and high selectivity.

functionalized

Scheme 1.

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Table 1.

Initial concentration of the

Spiked samples

Measured

(ng/mL)

amounts (ng/mL)

1

3

RSD (%)

Recovery (%)

2.013

4.76

102.9

3.932

2.01

98.26

serum (ng/mL)

0.984