AuCuxO-Embedded Mesoporous CeO2 Nanocomposites as a Signal

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Biological and Medical Applications of Materials and Interfaces

AuCuxO Embedded Mesoporous CeO2 Nanocomposites as Signal Probe for Electrochemical Sensitive Detection of Amyloid-beta protein Zengqiang Gao, Yueyun Li, Chunyan Zhang, Shuan Zhang, Yilei Jia, Faying Li, Hui Ding, Xinjin Li, Zhiwei Chen, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b01445 • Publication Date (Web): 11 Mar 2019 Downloaded from http://pubs.acs.org on March 18, 2019

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ACS Applied Materials & Interfaces

AuCuxO Embedded Mesoporous CeO2 Nanocomposites as Signal Probe for Electrochemical Sensitive Detection of Amyloid-Beta Protein Zengqiang Gao,†,‡ Yueyun Li,*, † Chunyan Zhang,† Shuan Zhang,† Yilei Jia,† Faying Li,‡ Hui Ding,‡ Xinjin Li,† Zhiwei Chen,♯ and Qin Wei*, ‡ † School of Chemistry and Chemical Engineering, Shandong University of Technology, Zibo 255049, P. R. China ‡ Key Laboratory of Interfacial Reaction & Sensing Analysis in Universities of Shandong, School of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022, P. R. China ♯ School of Life Sciences, Shandong University of Technology, Zibo 255049, P. R.

China

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ABSTRACT: A sandwich-type electrochemical immunosensor for detecting Amyloid-beta protein (Aβ) was fabricated based on Au NPs functionalized reduced graphene oxide (Au@rGO) as an effective sensing platform and AuCuxO embedded mesoporous CeO2 (AuCuxO@m-CeO2) nanocomposites as the catalytic matrix. The AuCuxO@m-CeO2 composites were obtained by adjusting the amount of m-CeO2 in reaction to expose enormous active sites. And AuCuxO@m-CeO2 was applied as a matrix to immobilize antibody by forming bridged bonds between m-CeO2 and carboxyl functional groups of antibody without additional agents. Furthermore, AuCuxO with prominent catalytic activities dramatically improved the performance of the fabricated immunosensor. And the morphology, structure, and electronic state of the surface were characterized by SEM, XRD, TEM, and XPS. In addition, the immunosensor demonstrated a wide linear range of 100 fg mL-1 to 10 ng mL-1. This study may provide a way for sensitively detecting various biomarkers.

KEYWORDS:

AuCuxO

composite,

mesoporous

sandwich-type immunosensor, Amyloid-beta protein


CeO2,

Au@rGO,

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1.INTRODUCTION Alzheimer’s Disease (AD) is a chronic neurodegenerative disease, which can affect cognitive competence and learning capacity.1 And the number of the affected people is remarkably increasing worldwide.2-3 As indicators of the disease state, biomarkers play a crucial role in early diagnosis, prognosis or response to treatment of cancer.4 Advances in therapeutic strategies for the AD and the development of the condition would dramatically relieve the global burden.5 Amyloid-beta protein is also referred to be Aβ, which consists of 36-43 amino acids and is the key component of the amyloid plaques in the brains of Alzheimer patients.6 And Aβ is also discovered to be implicated in biological regulation and psychological mechanisms.7-10 In addition, Aβ levels have been deemed to be connected with a number of cancers.11-12 Therefore, constructing a sensitive biosensing platform for Aβ is of significance in early diagnosis and treatment of the disease. As the advanced nanotechnology develops, many nanomaterials with marvelous catalytic activity have been introduced into electrochemical immunosensor to improve analytical performance by catalyzing the corresponding substrates, such as metal oxides,13 silicon nanoparticles,14 quantum dots,15 and magnetic nanomaterial.16 In particular, copper-based nanoparticles with the advantages of affordable price, abundant reserves, and unique electrochemical properties are of important materials in applications of catalysts, printable electronics, solar-energy conversion, and antimicrobial



agents.17-18 More importantly, the CuxO coupling CuO with Cu2O can exhibit excellent catalytic properties. As an efficient catalyst, CuxO has been confirmed that it has remarkable catalytic activities and superior selectivity towards H2O2.19 In addition, Au in the alloy obviously decreases the activation energy, making the reaction easier to occur.20 In the past years, more and more researches have been carried out for the synthesis of materials with porous nanostructures thanks to the large specific surface, good accessibility of the pores, and fast ion diffusion.21 Mesoporous CeO2 (m-CeO2) was applied as a carrier to prevent the metal composite from aggregation and migration, thanks to the uniform well-defined size distribution.22-23 Besides, m-CeO2 could immobilize abundant antibodies by forming bridged bonds.24-25 Finally, the developed immunosensor illustrated a wide linear range from 100 fg mL-1 to 10 ng mL-1, opening up a new way for the determination of biomarker. 2. EXPERIMENTAL SECTION 2.1 Synthesis of Au@rGO. In brief, 40 mL of 0.5 mg mL-1 GO was formed by ultrasonication. After several hours, 4 mL of 1.74 mM thionine and 500 μL of 2 % HAuCl4·4H2O solutions were injected. Then the suspension was stirred vigorously overnight at room temperature. The mixture was centrifuged and cleaning with double-distilled water, and the precipitation was left to dry by the vacuum freeze dryer.


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2.2 Synthesis of Ab2-AuCuxO@m-CeO2. The m-CeO2 nanospheres were synthesized on the basis of the reported method.26 Following that, 3 mg of m-CeO2 was dispersed in 20 mL of water under ultrasonication. Then, 200 μL of 2 % HAuCl4 solution and 400 μL of 1 % sodium citrate solution was subsequently added under vigorous stirring. After 5 min, 0.05 mmol of Cu(CH3COO)2·H2O was dissolved in the previous solution. And then 600 μL of 1 % sodium citrate solution containing 0.075 % NaBH4 was injected. While the mixture was stirred for 4 hours without interruption, the mixture was selected by centrifugation at 8000 rpm for 5 min. The Ab2-AuCuxO@m-CeO2 bio-conjugates were prepared as followed: Two mg of product was dispersed in 1.0 mL of PBS. Then 1 mL of 10 μg/mL Ab2 solution was added in the above solution, gently shocking for 12 h at 4 °C, making enough Ab2 immobilized on AuCuxO@m-CeO2. Following that, 100 μL of 1% BSA solution was added and shook for several hours. Finally, the product was centrifuged and separated. The Ab2-AuCuxO@m-CeO2 bioconjugate was dispersed in 1.0 mL PBS solution and placed at 4 °C.



Figure 1. Schematic Illustration for the fabrication of the proposed immunosensor. 2.3 Fabrication of the Sensing Platform. As shown in Figure 1, the GCE was firstly pretreated to be a mirror finish. After that, the pretreated GCE was decorated on 6 μL of 1.5 mg/mL Au@rGO suspension (Au@rGO/GCE). Then 6 μL of Ab1 solution was incubated onto the GCE (Ab1/Au@rGO/GCE), incubating at 4 °C for 12 h. After washed by PBS, the modified electrode was incubated for 40 min with BSA (BSA/Ab1/Au@rGO/GCE) to block nonspecific binding sites. After the excess BSA was removed with PBS, the modified GCE was incubated in 6 μL of antigen (Ag) for 1 h. Eventually, the GCE was incubated with 6 μL of Ab2-AuCuxO@m-CeO2 bioconjugates for 1 h and rinsed with PBS and ready for further measurement.


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3. RESULTS AND DISCUSSION 3.1 Characterization of Au@rGO. The successful synthesis of GO and Au@rGO was characterized by SEM, XRD, Raman spectra, and so on. As shown in Figure 2A, the GO sheets were composed of wrinkles and folds. In contrast, Au nanoparticles were uniformly distributed on rGO in Figure 2B, and the diameters distribution of Au calculated by simple software of Nano Measurer was 14 nm in Figure S1. The structural trends have been further proved by the XRD data and Raman spectra. As demonstrated in Figure 2C, the XRD data at a 2θ value of 9.8° corresponded to the (002) plane of GO with an interplanar distance of 9.06 Å. Compared with the parent GO, a broad peak of the disordered structure was observed at 21.6°, because the oxygen-containing groups were removed from GO and the layered rGO was formed. And the presence of diffraction peaks at 38.1 and 44.4° corresponded to (111) and (200) diffraction planes of gold (JCPDS NO. 04-0784). Raman spectra have been used to corroborate the conversion of GO to Au@rGO. As depicted in Figure 2D, the D-band was connected with the double resonance Raman scattering process at 1346 cm−1 and the G-band appearing at 1596 cm-1 due to the E12g symmetry of the sp2 bonding confirmed the GO was synthesized successfully.27 The D and G-bands in the Au@rGO compounds were respectively noticed at 1351 and 1601 cm-1. And the ID/IG ratios of GO and Au@rGO were 0.93 and 0.96, suggesting that defects in Au@rGO were enhancing.28 Moreover, the chemical conjugation of GO to Au@rGO was also



measured by FTIR spectroscopy and UV-vis absorption spectra shown in the supporting information.

Figure 2. SEM image of GO (A) and Au@rGO (B); XRD pattern (C) and Raman spectra (D) of GO (Top) and Au@rGO (Bottom). 3.2 Characterizations of AuCuxO@m-CeO2. As illustrated in Figure 3A, the m-CeO2 exhibited uniform-sphere morphologies with the average size of 120 nm. In addition, elemental atomic contents of Ce and O of m-CeO2 measured by EDX spectrum in Figure 3B are 29.34% and 70.66%. Obtained from Brunauer-Emmett-Teller methods (Figure 3C), the specific surface area of m-CeO2 was calculated to be 1386 m2/g, with the average pore size of 4.11 nm (BJH method, inset). The energy-dispersive X-ray (EDX) mapping in


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Figure 3E confirmed that Au and Cu elements were evenly distributed on m-CeO2. And a large number of lattice defects were found on the surface of the AuCuxO@m-CeO2 in Figure 3F, which may act as electron traps and accelerate the rate of catalytic reaction. Both the fast Fourier transformation diffractogram (Figure 3G) and configuration model (Figure 3H) of the AuCuxO@m-CeO2 structure were confirmed that.29 From XRD pattern in Figure S3, all peaks can be fitted to the pure face-centered cubic (fcc) CeO2 (JCPDS NO. 65-2075). Compared with the XRD pattern of m-CeO2, the additional XRD peaks appeared at 38.4°, 44.2°, 65.1°, and 78.1° can be designated to Au (111), (200), (220), and (311) planes, respectively. Nevertheless, no obvious diffraction plane of Cu was found due to the interference of the glass phase. Meanwhile, the structure of m-CeO2 was not destroyed. In addition, the morphology evolution of final products in the system was investigated in Figure S4. The disordered networks were obtained without m-CeO2. When m-CeO2 was added, the nanoparticles were formed and assembled on m-CeO2. It can be contributed to the unusual merits: (1) the large surface area and inherent porosity of m-CeO2 can supply amorous active sites for the growth of AuCu2O particles.; (2) m-CeO2 acted as the template effectively prevented AuCu2O nanoparticles from aggregating during the growth process.



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Figure 3. SEM image (A), EDX spectrum (B), and N2 adsorption-desorption curves image

of m-CeO2 (corresponding pore size distribution, inset) (C); TEM (D)

and

Energy-dispersive

X-ray

(EDX)

mapping

(E)

of

AuCuxO@m-CeO2; High-resolution TEM images of AuCuxO@m-CeO2 (F), corresponding fast Fourier transformation diffractogram (G) and configuration model (H) . In this work, XPS tests were performed to further investigate the surface electronic state and composition of AuCuxO@m-CeO2. As shown in Figure S5, the survey spectrum further certified the presence of Au, Cu, Ce, and O elements. As shown in the high-resolution Ce XPS spectrum (Figure S6A), the Ce4+ and Ce3+ chemical species with different peak were confirmed by Gaussian distributions.25 The binding energy peaks at 882.7, 899.4, 898.3, 901.2, 907.7, and 917.1 eV represented Ce4+, and peaks at 881.5, 885.3,


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898.9, and 903.6 eV belonged to Ce3+. The Ce3+/Ce4+ content ratio was 0.70. The XPS spectra peak of O 1s in the surfaces of AuCuxO@m-CeO2 were investigated in Figure S6B. The Oa was attributed to the defect oxide or hydroxyl-like group and the Ol was lattice oxygen, which was more stable than the former. The binding energy of Oa and Ol were about 531.5 and 529.5 eV, respectively. The Oa/Ol ratio representing the surface oxygen vacancy change was

1.15,

illustrating

AuCuxO@m-CeO2

possessed

higher

surface

chemisorbed oxygen. As shown in Figure S6C, the binding energy peaks at 83.8  and 87.5 eV for Au 4f7 and Au 4f5 doublet were corresponding to the standard binding energy values of Au0 state.30 As can be seen in Figure S6D, the doublet at binding energy of 932.7 and 952.8 eV belonged to Cu 2p3 and Cu 2p1, respectively.18,

31

The two peaks at binding energy of 932.66 and

952.45 eV were attributed to Cu 2p3/2 and Cu 2p1/2 of CuO.32 The higher peaks at 933.16 and 953.23 eV were the characteristic peaks of Cu2O. The estimated content ratio of CuO to Cu2O based on the XPS peak area was approximately 0.89, demonstrating that Cu+ species took a larger proportion in CuxO composite. And the detectable satellite peak at 943.64 eV further manifested the existence of CuO. The results above further confirmed that the grafted Cu species involved in Cu+ and Cu2+ compounds. 3.3 Electrocatalytic Activities towards Reducing H2O2. Catalytic activities of Au@rGO and AuCuxO@m-CeO2 towards electrochemical H2O2 reduction were evaluated by using cyclic voltammetry (CV) (Figure S7, Supporting



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Information). Both GCE and Au@rGO modified surfaces towards the reduction of H2O2 showed a little current, illustrating that Au@rGO possessed poor

catalytic

performance

for

H2O2.

Crucially,

the

response

of

AuCuxO@m-CeO2 in PBS buffer in presence of H2O2 is higher than Au@rGO in PBS buffer containing H2O2 and the AuCuxO@m-CeO2 exhibited two peaks at approximately 0 V and -0.4 V, illustrating that the admirable electro-catalytic activities towards H2O2 may be accomplished in two steps due to different Cu species. And the peak of -0.4V can be attributed to Cu2O and the other at 0 V may be attributed to CuO.33

Figure 4. (A) Linear sweep voltammograms (LSV) in PBS solution (pH 7.4) containing 10 mM H2O2 with a scan rate of 100 mV s-1 and (B) i-t curves at -0.4 V of m-CeO2, Au@m-CeO2, Cu@m-CeO2, and AuCuxO@m-CeO2. Catalytic

activities

of

m-CeO2,

Au@m-CeO2,

Cu@m-CeO2,

and

AuCuxO@m-CeO2 on electrode were also evaluated using voltammetric methods (Figure S6, Supporting Information). The current signals among them in LSV (Figure 4A) showed two onsets at the potential of about 0 and


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-0.2 V and reached a limiting current at potentials below -0.4 V. Obviously, AuCuxO@m-CeO2 exhibited the largest peak current, which was more than three times as big as that of Cu@m-CeO2. And the catalytic activities for different electrodes via current-time curves (i-t) in Figure 4B followed the order: AuCuxO@m-CeO2/GCE > Cu@m-CeO2/GCE > Au@m-CeO2/GCE > m-CeO2/GCE. The above results confirmed that AuCuxO@m-CeO2 had superior catalytic activities. 3.4 The Mechanism of the Reaction. The AuCuxO@m-CeO2 produced a cathode current with the presence of H2O2. Reciprocal transformation of copper ions possessed highly redox active activities, which can produce hydroxyl radical by the following Fenton-like reaction.34-35 Cu2+ nanoparticles reacted with H2O2 transferred into redox active copper ions (Cu+). Meanwhile, Cu+ was oxidized to Cu2+. Following by reciprocal transformation, the m-CeO2 with high oxygen vacancies could react with the radical to generate O2.36 3.5 Characterization of Immunosensor Interface. The cyclic voltammetry (CV) was implemented to investigate the interface properties and the curves are shown in Figure 5A. The bare GCE exhibited a well-defined redox peak (curve a). And the current increased after modifying conductive Au@rGO composites (curve b), illustrating that the Au@rGO had good electrical conductivity.

But

when

Ab1,

BSA,

antigen

(Ag,

10

ng/mL),

and

Ab2-AuCuxO@m-CeO2 were successively assembled on the GCE, the corresponding peak currents (curve c, d, e, and f) were gradually decreased,



which may be attributed to the hindrance of protein. Furthermore, the electrode interface properties were further studied by using EIS through monitoring the change of electrode impedance on the Nyquist plot.37 As illustrated in Figure 5B, the charge transfer resistance values (Rct) of GCE (curve a) was 68.78 Ω. After Au@rGO was decorated on GCE, a smaller Rct value (curve b) was observed (40.26 Ω), thanks to the good electrical conductivity of Au@rGO. There were found to be 139.36, 275.1, 643.4, and 1130 Ω for Rct when Ab1, BSA, Ag, and Ab2-AuCuxO@m-CeO2 were immobilized in the sequence onto modified GCE surface. Both CV and EIS results proved that immunosensor was successfully prepared.

Figure 5．Cyclic voltammetry (A) and EIS responses (B) of: (a) bare GCE; (b) Au@rGO/GCE; (c) Ab1/ Au@rGO/GCE; (d) BSA/ Ab1/ Au@rGO/GCE; (e) Ag/BSA/Ab1/Au@rGO/GCE; (f) Ab2-AuCuxO@m-CeO2/ Ag/BSA/Ab1/Au@rGO /GCE. 3.6 Performance of the Immunosensor. The i-t responses for different concentrations of Aβ were investigated under optimized conditions and the


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results were shown in Figure 6. Obviously, the current response increased with the elevated concentration of Aβ and the current response presented proportionally to the logarithm of the concentration of Aβ ranging from 100 fg·mL−1 to 10 ng·mL−1. The linear equation could be displayed as Ip = 25.2678 log cAβ - 112.2027 with the correlation coefficient R2 = 0.9964. Besides, the limit of detection is 36 fg mL-1. Compared with previous literature listed in Table 1, the developed immunosensor exhibited higher sensitivity. That could attribute to the excellent performance of the Au@rGO and AuCuxO@m-CeO2 composites, which had an irreplaceable effect on satisfactory detection.

Figure 6. Current response (A) and calibration curve (B) of proposed immunosensor with 10 μL 10 mM H2O2 in 10 mL PBS solution (pH 6.24) for the different content of Aβ (from 100 fg/mL to 10 ng/mL, a → f). Error bars = SD (n = 3).



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Table 1. The performance comparison of the proposed immunosensor for Aβ detection. Detection method

Electrochemic al immunosensor

Fluorescence

Electrochemic al immunosensor

Fabrication strategy

Linear

Detection Referen

range

limit

AuNPs electrodeposited mercaptopropionic acid 10 - 1.0 × 5.2 38

103 pg·mL-1 pg·mL-1

self-assembled monolayer Streptavidin-QDs

23 - 456.2 7.6 39

pg·mL-1 Screen-printed

carbon

electrode nanostructured with gold nanoparticles

pg·mL-1

5.0 × 102 5.0 ×

105

pg·mL-1

Electrochemilu Ceria doped zinc oxide 8.0 × 10-2 -minescence

nanoflowers

and 1.0 ×

immunosensor electrodeposited gold Electrochemic

Anodic aluminum oxide

al impedance membrane, spectroscopy

ces

nanoparticles

gold

pg·mL-1

105

100 40

pg·mL-1

0.052 41

pg·mL-1

1.0 - 1.0 × 1.0 42



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Colorimetric

AuNPs@C/N-Ab(1-42)

sandwich

7.5 - 350

immunosensor

nM

2.3 nM

43

system photoelectroch Mn:CdSe

to 0.2 ~ 5.0× 0.068

emical sensor

Bi2WO6/CdS

Electrochemic

AuCuxO

al

mesoporous

immunosensor Au@rGO

104 embedded CeO2,

44

pg·mL-1

0.10 - 1.0× 0.036

This


work

The reproducibility was a crucial factor for real clinical application of proposed immunosensors.45,46 Figure S8A showed that five immunosensors were constructed and measured under the same conditions. And there were no significant differences among the current response.47, 48 The specificity of the proposed immunosensor was also evaluated with the interferents of insulin, prostate specific antigen (PSA), alpha fetal protein (AFP). As diagramed in Figure S8B, the relative standard deviation of the current signals was less than 5%, illustrating that the immunosensor had favorable specificity. The long-term stability was also measured every three days. As depicted in Figure S8C, the current retained 92.5 %, 92.7 %, and 93.2 % of their initial current signals after 21 days, indicating that the proposed immunosensors



possessed good stability. And CV curves (50 cycles) in Figure S8D further illustrated that the signal label had good stability (5.43%). 3.7 Determination of Aβ. To assess the promising potential of the proposed immunosensor, the proposed electrochemical immunosensors were used to determine Aβ in artificial cerebrospinal. The recovery shown in Table S1 varying from 94 % to 109 % suggested that the immunosensor had a promising potential in the detection of Aβ. 4. CONCLUSIONS The immunosensor with preferable sensitivity, specificity, and stability was successfully fabricated on the basis of AuCuxO embedded mesoporous CeO2 for Aβ detection. Thanks to the large surface-to-volume rate and rich porosity of mesoporous CeO2, the size of AuCuxO composites tended to be smaller, making more active sits exposed, which exhibited better catalytic activity towards H2O2. Furthermore, Au NPs functionalized reduced graphene oxide with good biocompatibility was implemented to develop a sensitivity enhancement strategy for immunosensors. Considering the commendable properties, there was a potential application for the determination of AD biomarker in bioassays or other biosensors. ASSOCIATED CONTENT Supporting Information. The following files are available free of charge.


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Figure S1. FT-IR and UV-vis spectra of the GO and Au@rGO; Figure S2. XRD pattern of m-CeO2 and AuCuxO@m-CeO2; Figure S3. TEM images of the products; Figure S4. XPS survey spectrum of AuCuxO@m-CeO2; Figure S5. Cyclic voltammograms (CV) of the different electrodes (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y.L.) *E-mail: [email protected] (Q.W.) Author Contributions All authors have given approval to the final version of the manuscript. ACKNOWLEDGMENT This study was supported by the Key Research and Development Program of Shandong Province (No. 2018GSF120001, 2018GNC110038), National Natural Science Foundation of China (No. 21575079), the National Key Scientific Instrument and Equipment Development Project of China (No. 21627809). Notes The authors have no competing financial interest. REFERENCES



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Table of Contents Graphic


AuCuxO-Embedded Mesoporous CeO2 Nanocomposites as a Signal

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