Dual Quenching Electrochemiluminescence Strategy Based on 3D

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Dual Quenching Electrochemiluminescence Strategy Based on 3D Metal-organic Frameworks for Ultrasensitive Detection of Amyloid-# Guanhui Zhao, Yaoguang Wang, Xiaojian Li, Qi Yue, Xue Dong, Bin Du, Wei Cao, and Qin Wei Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04332 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Analytical Chemistry

Dual Quenching Electrochemiluminescence Strategy Based on 3D Metal-organic Frameworks for Ultrasensitive Detection of Amyloid‑β

Guanhui Zhao, Yaoguang Wang, Xiaojian Li, Qi Yue, Xue Dong, Bin Du, Wei Cao*, Qin Wei*

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

* Corresponding author E-mail: [email protected] (W. Cao). [email protected] (Q. Wei). Tel.: +86 53182767890 Fax: +86 53187161600

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Analytical Chemistry 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

ABSTRACT We have proposed a dual quenching electrochemiluminescence (ECL) strategy which based on Ru(bpy)32+ as chromophores caged in 3D Zn-oxalate metal-organic frameworks (Ru(bpy)32+/Zn-oxalate MOFs) for ultrasensitive detection of amyloid‑β (Aβ). The three-dimensional chromophore connectivity in Zn-oxalate MOFs provided a network for rapid excited state energy transfer migration among Ru(bpy)32+ units which shielded the chromophores from solvent molecules and led to a high energy Ru emission efficiency. In addition, we found that both Au nanoparticles and NiFe-based nanocube MOFs could contribute to the reduction of the ECL intensity of chromophore. And the ECL emission spectra of 3D Ru(bpy)32+/Zn-oxalate MOFs overlapped appropriately with the ultraviolet visible (UV-vis) absorption spectra of Au@NiFeMOFs composites, which could trigger the resonance energy transfer (RET) behavior between Ru(bpy)32+/Zn-oxalate MOFs (donor) and Au@NiFe-MOFs (acceptor), achieving the dual quenching effect of Ru(bpy)32+ encapsulated in 3D Zn-oxalate MOFs and significantly boosting the sensitivity of the Aβ-detection immunosensor. In order to examine the clinical practicability, we have applied it to verify the content of Aβ solution ranging from 100 fg mL-1 to 50 ng mL-1 and obtained the calibration cure with high correlation coefficient, along with the low limit of detection of 13.8 fg mL-1. Above all, this work demonstrated an approach of constructing dual quenching effect ECL immunosensors in whole 3D MOFs system and its application in ECL detection methodology. Keywords: Dual quenching electrochemiluminescence; Ru(bpy)32+; 3D Zn-oxalate


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metal-organic frameworks; Au@NiFe nanocube; Amyloid‑β detection



INTRODUCTION Alzheimer’s disease (AD) as a progressive irreversible neurological disorder, which could cause memory loss, behavior disability, and even death, has extremely threatened human health and reduced the life quality of many middle-aged and old people seriously nowadays.1,2 On the basis of AD study, the aggregation of amyloid‑β (Aβ) was testified to be the pathogenesis of AD due to its extremely strong neurotoxicity.3,4 Therefore, Aβ, which is a 39-43 amino acid peptide fragment deriving from amyloid precursor protein, was used as one of the main diagnostic biomarkers and therapeutic targets of AD.3,5,6 Hence, it was very meaningful to explore various efficient methods to determine the concentration of Aβ at the early stages of AD accurately and sensitively. Specifically, electrochemiluminescence (ECL) immunosensor as an ECL immune-modified platform has drawn tremendous attention gradually due to its low background interference, simplified instrument operation, and high sensitivity, etc.7-9 According to the previously reported literatures, multitudinous materials were used in the ECL strategies to determinate Aβ such as ceria doped zinc oxide nanoflowers, functionalized-luminol, and silver nanoclusters/titanium oxide nanomaterials.5,7 And among various ECL researches, Ru(bpy)32+ was one of the most frequently-used luminophor in the ECL system on account of its stable luminous efficiency, less dosage, and pellucid luminescence mechanism.8,10,11 However, it was difficult to apply Ru(bpy)32+ to the solid-state sensing platform directly because of its excellent water solubility.12 And many methods about how to immobilize Ru(bpy)32+ in the water


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environment have been explored by the researchers.13,14 The good news was that using MOFs materials to encapsulate Ru(bpy)32+ has become into focus due to its porosity, adjustable aperture, larger specific surface area, and homogeneous appearance.15,16 In a variety of MOFs materials, the three-dimensional (3D) oxalate-MOFs has been applied increasingly to load Ru(bpy)32+ in the ECL immunosensors.17,18 3D oxalate-MOFs microcrystals with chromophoric building blocks possess large enough dimensions to absorb a high fraction of light and yet micro enough for the excited states to be delivered by energy transfer processes to a redox reaction center at the MOFs/solution interface.15 In this work, the Ru(bpy)32+ cations were entrapped in anionic three-dimensional Zn-oxalate MOFs possessing cage structures with the purposes of increasing the dimensionality of energy transfer pathways while maintaining the electronic structure of the Ru(bpy)32+ chromophore unperturbed.19 And the electronic structures and energy of the D3 symmetric Ru(bpy)32+ was much more orderly compared with those previously reported photoactive MOFs, which made the Ru(bpy)32+ exhibit long-lived luminescence lifetime and high quantum yield.20,21 Furthermore, since the resonance energy transfer (RET) behavior could occur between Au@NiFe MOFs (acceptor) and Ru(bpy)32+/Zn-oxalate MOFs (donor), the Au@NiFe MOFs with the nanocubic morphology were applied as the ECL quencher of Ru(bpy)32+ encapsulated in Zn-oxalate MOFs in the proposed immunosensor. Numerous studies have confirmed that the synergy of nickel and iron could modify electronic structure and improve the electrical conductivity.22,23 The unique porous structure of NiFe-based MOFs could also provide more active sites and shorten the



mass transport distance effectively.23-25 It was worth mentioning that the UV-vis absorption spectrum of NiFe-based MOFs and Au@NiFe MOFs all overlapped with the ECL emission spectra of Ru(bpy)32+/Zn-oxalate MOFs, respectively. Thus, both NiFe-based MOFs and Au@NiFe MOFs could quench the luminous efficiency of Ru(bpy)32+/Zn-oxalate MOFs.26,27 Nevertheless, due that Au nanoparticles have an absorption peak at 550 nm, the UV-vis absorption spectrum of Au@NiFe MOFs overlapped more with the ECL emission spectrum of Ru(bpy)32+/Zn-oxalate MOFs compared with NiFe-based MOFs. Thereby, the dual quenching effect of ECL efficiency of Ru(bpy)32+/Zn-oxalate complex could be achieved and the sensitivity of the proposed immunosensor could be significantly improved . In this way, a dual quenching ECL immunosensor which was based on Ru(bpy)32+/Zn-oxalate MOFs as luminophor and Au@NiFe-MOFs as the dual-quencher has been constructed for the ultrasensitive detection of Aβ. And this also could provide a novel approach to determine the protein toxins in the clinical medicine.

EXPERIMENTAL SECTION Preparation of Ru(bpy)32+/Zn-oxalate MOFs. The synthesis of Zn-oxalate MOFs was based on previous report

28

with a little improvement. Typically, 15 mg of

Ru(bpy)3Cl2·6H2O and 100 mg of Zn(NO3)2 were added into 45 mL mixture of DMF and water (2:1) with 300 μL of oxalic acid (0.75 mol L-1 in DMF) and 3 mL of HCl (3 mol L-1). After ultrasonic vibrating for 5 min, the mixture was transferred to the roundbottom flask and reflux at 60 ℃ for 24 h. The obtained orange-red precipitate was washed with DMF, ethanol and ultrapure water two times respectively and dried in


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vacuum at 50 ℃ overnight. Preparation of the Monomeric, oligomeric, and fibrous Aβ. The Aβ monomers, oligomers (AβO), and fibrils (AβF) were prepared according to previous literatures.2933

Briefly, lyophilized Aβ peptide was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol

and sonicated for 10 min to form the monomers. Afterwards, the resulting products were reconstituted in NaOH solution, diluted by PBS solution (pH 7.4), and incubated for 16 h at room temperature to form AβO. Similarly, the same process as above and incubated for 24 h at 37 ℃ to form AβF. A centrifugation step was needed at 14000 r for 15 min to remove any insoluble aggregates. Preparation of Ru(bpy)32+/Zn-oxalate MOFs/Ab1 Bioconjugate. The synthetic process of Zn-oxalate MOFs/Ab1 bioconjugate was shown in Scheme 1A. Primarily, in order to activate the carboxyl on the surface of the Zn-oxalate MOFs, 2 mL of 1 mg mL-1 Zn-oxalate MOFs which dispersed in PBS solution (pH 7.4) was mixed with EDC (20 mmol) and NHS (10 mmol) for 2 h at 4℃. After that, 500 μL of 1 μg mL-1 antibodyAβ (Ab1) was added into the above mixture and continued stirring for another 12 h to bind on the surface of Zn-oxalate MOFs via the amide reaction. The generated Znoxalate MOFs/Ab1 bioconjugate was collected by centrifugation and washed with PBS solution (pH 7.4) for once, ultimately dispersed in 1 mL of PBS solution (pH 7.4) and stored at 4℃ for further use. Synthesis of Au@NiFe MOFs. Primarily, the NiFe-based nanoporous MOFs were prepared through a coprecipitation method.23 Typically, nickel chloride hexahydrate (0.6 mmol) and tri-sodium citrate dehydrate (0.9 mmol) were dissolved in 20 mL of



deionized water, and 0.4 mmol of potassium ferricyanide was dissolved in 20 mL of deionized water. After that, the above two solutions were mixed together under continuous magnetic stirring followed by aging at room temperature for 10 h. The earthy-yellow precipitate was collected by centrifugation and washed with deionized water and ethanol for two times, respectively. The obtained product was dried overnight at 50℃. Au@NiFe MOFs were obtained by in-situ reduction of gold nanoparticles on the surface of NiFe-based nanoporous MOFs.17 Typically, 50 mg of the prepared NiFebased nanoporous MOFs and 3 mL of 2% HAuCl4 were dispersed in 20 mL of deionized water under magnetic stirring for 5 min. Afterwards, 7 mg of PVP were added into the solution for another 5 min of magnetic stirring to prevent the agglomeration of gold nanoparticles. Then 5 mL of 5 mol L-1 sodium citrate solution and a drop of sodium borohydride solution were added to the above solution as reductant and continuing stirring for 12 h. The generated atropurpureus precipitation was washed with deionized water until the supernatant was colorless dried overnight at 50℃. Preparation of Ab2-Au@NiFe MOFs Bioconjugate. The preparation of Ab2Au@NiFe MOFs bioconjugate was demonstrated in Scheme 1B. Firstly, 2 mg of Au@NiFe MOFs was dispersed in 500 μL of pH 7.4 PBS solution and mixed with EDC (20 mmol) and NHS (10 mmol) for 2 h at 4℃. After removing the excess crosslinker, the mixture mixed with 500 μL of 1 μg mL-1 Ab2 under vibration at 4℃ for 24 h. The generated Ab2-Au@NiFe MOFs complex was gathered through centrifugation and washed with pH 7.4 PBS solution for once. After that, 150 μL of 1% BSA was mixed


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with the above product and vibrated at 4℃ for 6 h to block the non-specific active sites on the surface of Au@NiFe MOFs. The final product was rinsed with pH 7.4 PBS solution to wash away the unreacted BSA and dispersed in 1 mL pH 7.4 PBS solution (pH 7.4). Then it was stored at 4℃ for further use. Fabrication of the Dual Quenching ECL Immunosensor. The fabrication of the ECL immunosensor was shown in Scheme 1C. Prior to modification, the electrode (GCE) was polished to a mirror by using 1.0, 0.3, and 0.05 μm alumina slurry, followed by sonication in deionized water and ethanol. The electrode was rinsed thoroughly with deionized water and dried in N2 flow. Afterwards, 6 μL of 0.8 mg mL-1 Ru(bpy)32+/Znoxalate MOFs/Ab1 bioconjugate was directly immobilized onto the clear electrode surface, drying at 4℃. Subsequently, 3 μL of 1% BSA was dropped onto the surface of modified electrode and incubated at 4℃ for 2 h to block the non-specific active sites. Next, the superfluous BSA was washed away when the GCE surface was damp and dried. Then 5 μL of different concentrations of A β was modified onto the above electrode, incubating at 4℃ for 2 h to specific bind with Ab1. Subsequently, the unreacted Aβ was scoured off when the electrode surface was slightly wet and dried at 4℃. Eventually, 6 μL of 0.5 mg mL-1 Ab2-Au@NiFe MOFs bioconjugate was incubated onto the prepared electrode through specific binding and rinsed with pH 7.4 PBS solution thoroughly to remove the redundant antibody. The dual quenching MOFs ECL system has been constructed and stored at 4℃ for further use.

RESULTS AND DISCUSSION Morphology Characterization of Ru(bpy)32+/Zn-oxalate MOFs, NiFe-based MOFs



and Au@NiFe MOFs. Scanning electron microscope (SEM) images of Ru(bpy)32+/Znoxalate MOFs were depicted with different scales in Figure 1A and showed typical octahedral morphology. Besides, the energy dispersive spectroscopy (EDS) spectrum of Ru(bpy)32+/Zn-oxalate MOFs was obtained in Figure S1 and it could be observed that the major elements were Zn, Ru, C, and O, indicating that the Ru(bpy)32+ luminophor was encapsulated in Zn-MOFs successfully. Figure 1B and 1C were SEM and HRTEM images of NiFe-based MOFs, respectively, and it could be observed that the NiFe-based MOFs presented as homogeneous cubes with average diameter of 100 nm. As the auxiliary quench agent and the active sites bound with antibodies, Au nanoparticles were evenly distributed on the surface of NiFe-based MOFs by the method of in situ reduction of growth as Figure 1D and 1E shown. In addition, the acquired EDAX mapping images were shown in Figure 1F, which showed that the elements of Ni, Fe were primary components of NiFe-basded MOFs and Au nanoparticles were reduced to the surface of NiFe-based MOFs successfully. Moreover, the powder X-ray diffraction (PXRD) patterns, the fourier transform infrared spectroscopy (FT-IR) spectrogram and the EDS were acquired to further explore the characters of Ru(bpy)32+/Zn-oxalate MOFs, NiFe-based MOFs, and Au@NiFe MOFs. As shown in figure 2A, the PXRD patterns demonstrated that Ru(bpy)32+/Zn-oxalate MOFs exhibited well crystallinity and identified to the simulated pattern of the reported single crystal structure.15 Ru(bpy)32+/Zn-oxalate MOFs could bind with antibodies through the cross-linking agents since there are plentiful carboxyl groups on the surface of Ru(bpy)32+/Zn-oxalate MOFs as


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characterized in Figure 2B. The FT-IR absorption peak at 1322 cm-1 was the C-O vibration of the single bond. The characteristic absorption peak of C=O double bond vibratory expansion appeared at 1625 cm-1. In addition, the characteristic absorption peak of O-H stretching vibration was discovered at 3415 cm-1.13,17,34 Figure 2C showed the PXRD spectrum of NiFe-based MOFs and Au@NiFe MOFs. It could be observed that the synthesized NiFe-based MOFs exhibited typical diffraction peaks of Fe2O2CO3 (JCPDS card 33-0665) and Fe0.93Ni0.056 (JCPDS card 44-1088).23 And it could also indicated the presence of carboxyl groups over the surface of Au@NiFe MOFs as Figure S2 shown, which could make the Au@NiFe MOFs link with more Ab2 to improve the sensitivity of the dual quenching ECL immunosensor. Moreover, the crystallinity did not change when the Au nanoparticles were grown in situ on the surface of NiFe-based MOFs. The major elements of Au@NiFe MOFs have been analyzed by EDS spectrum and atomic percentages as Figure 2D shown. Ni, Fe, Au, C, and N were found and used for the constitution of Au@NiFe MOFs as the main elements. The surface chemical compositions and elemental valence states of the NiFe-based MOFs material were assessed by X-ray photoelectron spectroscopy (XPS) measurements. The survey spectrum in Figure S2 obviously indicated the presence of Ni, Fe, and C, which were consistent with the elemental mapping images. For NiFebased MOFs, the high-resolution spectra of Fe 2p, Ni 2p, and C 1s were presented in Figure 3. In the Ni 2p region (Figure 3A), the binding energies appeared at 856.3 eV and 873.9 eV, revealing the formation of oxidized nickel species. The high-resolution spectrum of Fe 2p (Figure 3B) displayed two peaks located at 709.7 eV and 723.5 eV,



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confirming that the presence of the oxidation state of iron with Fe3+ as the primary existence form.23 Moreover, the high-resolution spectrum of C 1s (Figure 3C) exhibited one peak seated at 284.6 eV. Mechanism Investigation of the Dual Quenching MOFs System. Ru(bpy)32+ luminophore which was inseted in the Zn-oxalate MOFs displayed anodic emission light in the presence of co-reactant tripropylamine (TPA) at the voltage range from 0 to 1.3 V. Two kinds of speculations of the reaction mechanism were supposed in this Ru(bpy)32+-TPA ECL system.11,35,36 Route 1 showed one supposed ECL mechanism to generate excited-state Ru(bpy)32+, primarily TPA was directly oxidized to TPA•+ in the low potential ECL and deprotonated to TPA•. The TPA• radical species reduced Ru(bpy)32+ to form Ru(bpy)3+, which further reacted with the oxidative species TPA•+ to form Ru(bpy)32+*. Route 2 showed another ECL mechanism occurred at high potential. Firstly, Ru(bpy)32+ was oxidized to Ru(bpy)33+ directly on the electrode, which further reacted with the TPA• radical species to generate Ru(bpy)32+*. In addition, an alternative mechanism for generating Ru(bpy)32+* at the higher potential was raised as that Ru(bpy)33+ generated in Route 2 reacted with Ru(bpy)3+ generated in Route 1 to form Ru(bpy)32+*. Route 1 TPA – e

TPA•+

TPA•+ – H+

(1a)

TPA•

TPA• + Ru(bpy)32+ Ru(bpy)3+ + TPA•+

(1b) Ru(bpy)3+ + product Ru(bpy)32+* + product


(1c) (1d)

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Ru(bpy)32+*

Ru(bpy)32+ + hv

(1e)

Ru(bpy)33+

(2a)

Route 2 Ru(bpy)32+ – e TPA – e

TPA•+

TPA•+ – H+ Ru(bpy)33+ + TPA• Ru(bpy)32+*

(2b)

TPA•

(2c) Ru(bpy)32+*

Ru(bpy)32+ + hv

(2d) (2e)

To investigate the quenching effect of the Ru(bpy)32+/Zn-oxalate MOFs by NiFebased MOFs and Au nanoparticles, the UV-vis absorption, fluorescence emission, and ECL behavior of NiFe-based MOFs, Au@NiFe MOFs and Ru(bpy)32+/Zn-oxalate MOFs were explored. As shown in Figure 4A, the UV-vis absorption spectrum of NiFebased MOFs overlapped appropriately with the ECL emission spectrum of Ru(bpy)32+/Zn-oxalate MOFs, which triggered the quenching effect of Ru(bpy)32+/Znoxalate MOFs ECL because of the RET between Ru(bpy)32+/Zn-oxalate MOFs (donor) and NiFe-based MOFs (acceptor).37,38 After in situ reduction of Au nanoparticles on the surface of NiFe-based MOFs, the Au@NiFe MOFs compound exhibited a new UVvis absorption peak at 550 nm, which increased the spectrum overlap area of Ru(bpy)32+/Zn-oxalate MOFs and Au@NiFe MOFs and achieved a second quenching effect of Ru(bpy)32+ inlaid in Zn-oxalate MOFs. As a result, the quenching efficiency of Au@NiFe MOFs for Ru(bpy)32+/Zn-oxalate MOFs was higher than NiFe-based MOFs alone. Figure 4B was the fluorescence emission spectra of Ru(bpy)32+/Zn-oxalate MOFs



(a), the mixture of Ru(bpy)32+/Zn-oxalate MOFs and NiFe-based MOFs (b), and the mixture of Ru(bpy)32+/Zn-oxalate MOFs and Au@NiFe MOFs (c), respectively. It could be observed that the fluorescence emission intensity of Ru(bpy)32+/Zn-oxalate MOFs decreased when mixed with NiFe-based MOFs, and further decreased when mixed with Au@NiFe MOFs. Therefore, it could be concluded that NiFe-based MOFs could quench the emitting intensity of Ru(bpy)32+/Zn-oxalate MOFs, while the Au@NiFe MOFs compound could dual quench of the emitting intensity of Ru(bpy)32+/Zn-oxalate MOFs. Moreover, the ECL behaviors of Ru(bpy)32+/Zn-oxalate MOFs and the quenchers also were investigated. As shown in Figure 4C, the ECL signal response of the individual Ru(bpy)32+/Zn-oxalate MOFs was 14200 a.u., and the ECL intensity was reduced to almost half (7150 a.u.) when NiFe-based MOFs was dropped onto the surface of Ru(bpy)32+/Zn-oxalate MOFs/GCE. Meanwhile, when Au@NiFe MOFs was dropped onto the surface of Ru(bpy)32+/Zn-oxalate MOFs/GCE, the ECL response would be further reduced (6000 a.u.) compared with the curve b. Hence, the dual quenching effect of Au@NiFe MOFs on Ru(bpy)32+/Zn-oxalate MOFs ECL was confirmed again. Characterization of the Immunosensor. In order to demonstrate the modification process of each step for the electrode, the change of the ECL intensity of the proposed immunosensor was shown in Figure 5A. It could be observed that bare electrode has almost no ECL response in the presence of coreactant TPA. Whereas there was a very strong ECL response when Ru(bpy)32+/Zn-oxalate MOFs were modified onto the


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electrode (curve b). And the ECL intensity declined successively when dropped Ab1, BSA, and Aβ onto the modified electrode, respectively. This was because Ab1, BSA, and Aβ all were protein and could obstruct electron transfer seriously. The ECL intensity reduced to minimum after dispensed Ab2-Au@NiFe MOFs onto the modified electrode finally, which was because that the RET reaction occurred between Ru(bpy)32+/Zn-oxalate MOFs (donor) and Au@NiFe MOFs (acceptor) and caused the dual quenching effect on Ru(bpy)32+ ECL. It also has been proven that electrochemical impedance spectroscopy (EIS) was an useful way for the fabrication characterization of the immunosensor.39 As Figure 5B shown, the electron transfer resistance (Ret) of Ru(bpy)32+/Zn-oxalate MOFs/GCE (curve b) increased compared with that of the bare GCE (curve a), which indicated the successfully assembling of Ru(bpy)32+/Zn-oxalate MOFs. When Ab1 was joined with the Zn-oxalate MOFs through -NH2 and -COOH by crosslinking agent (EDS 40 mg, NHC 10 mg), the value of Ret increased significantly. After BSA, Aβ, and Ab2Au@NiFe MOFs were modified onto the above electrode, the value of Ret continued increasing. This may be due that BSA, Aβ, and Ab2-Au@NiFe MOFs all contained protein which would hinder the electron transfer greatly. Optimization of the Detection Conditions. The suitable environment of pH was crucial for the performance of the suggested immunosensor. After investigated the adaptability of the immunosensor to the peracidity environment, parlkaline environment, and neutral environment, the neutral environment (pH 7.4) was selected for the following study as shown in Figure 6A. TPA was the coreactant of Ru(bpy)32+



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encapsulated in the Zn-oxalate MOFs and the concentration of tripropylamine was optimized (Figure 6B). It could be observed that the ECL response achieved the strongest when the concentration of tripropylamine was 10 mmol L-1. In addition, although Ru(bpy)32+/Zn-oxalate MOFs displayed extraordinary ECL performance, too much of it would trigger the behavior of self-absorption which would shrinkage the ECL response.40 As shown in Figure 6C, the ECL signal increased when the concentration of Ru(bpy)32+/Zn-oxalate MOFs was less than 0.5 mg mL-1 and decreased when the concentration was greater than 0.5 mg mL-1. Therefore, 0.5 mg mL1

of Ru(bpy)32+/Zn-oxalate MOFs was used in the ECL system. The quantity of

Au@NiFe MOFs which as the acceptor of RET was also explored as shown in Figure 6D. The ECL intensity decreased to the lowest when the concentration of Au@NiFe MOFs labeled on the Ab2 was 0.8 mg mL-1, indicating that the RET achieved the highest efficiency. As a result, 0.8 mg mL-1 of Au@NiFe MOFs was chosen for further study. Performance of the ECL Immunosensor in Aβ Detection. Under the optimal experimental conditions, the analytical performance for the proposed immunosensor was investigated by incubating various concentrations of Aβ. The results were presented in Figure 7A. Bare GCE has hardly any ECL response (curve a) and an exceptionally strong ECL signal was obtained when modified Ru(bpy)32+/Zn-oxalate MOFs onto the bare GCE (curve k). The ECL intensity declined in turn with the increasing concentration of Aβ incubated onto the modified electrode (curve j

b).

Moreover, the ECL response of the proposed immunosensor exhibited an excellent linear relationship with the logarithm of Aβ concentration as shown in Figure 7B. The


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equation of linear regression was I = 5588.26 – 1188.20 lg c (I represented the ECL intensity and c represented the concentration of Aβ) and the correlation coefficient was 0.993. The estimated limit of detection (LOD) was 13.8 fg mL-1 (the calculation was shown in the supporting information),41 which exhibited better performance than that of many other reports as Table S1 shown, indicating the outstanding potentiality of the suggested immunosensor in bioassays. Furthermore, reproducibility, selectivity, and stability were also important indicators for assessing the analytical performance of a immunosensor.42 Under the optimized conditions, seven immunosensors were constructed in the same environment to determine Aβ simultaneously as shown in Figure 7C (RSD = 1.32%), indicating the superior reproducibility of the proposed immunosensor. Besides, in order to investigate the selectivity,43 the constructed immunosensor was used to determine the solution of human immunoglobulin (IgG), carcinoembryonic antigen (CEA), insulin, alpha fetoprotein (AFP), Aβ oligomers (AβO), Aβ fibrils (AβF), Aβ and mixture of them, respectively and the results were shown in Figure 7D. It could be observed that the ECL intensity was almost same as that of blank when the detecting system only contained the interferents of IgG, CEA, insulin, AFP, AβO and AβF respectively and significantly decreased when the detecting system contained Aβ, revealing that the suggested immunosensor was bound to Aβ specifically (three groups were implemented simultaneously). The stability was investigated by detecting different concentrations of Aβ for three circles and the consequences have been shown in Figure S3., indicating the favourable stability of the proposed immunosensor. Hence, all the excellent



performances could prop up that the constructed immunosensor has potential application in clinical analysis. Real Sample Analysis. In order to verify the clinical application of the proposed immunosensor, we applied it to determine the concentration of Aβ in the artificial cerebrospinal and testified the accuracy with standard addition method. The results were shown in Table S2. When the additive amount of Aβ was 0.005 ng mL-1, 0.1 ng mL-1, 5 ng mL-1, and 25 ng mL-1 respectively, the relative standard deviation (RSD) was less than 5% and the recovery was within the scope of 99% ~ 100.76%, illustrating the advantageous potential possibility of clinical application.

CONCLUSION In summary, a dual quenching ECL immunosensor has been proposed for the ultrasensitive detection of Aβ, which based Ru(bpy)32+ encapsulated in Zn-oxalate MOFs (Ru(bpy)32+/Zn-oxalate MOFs) as luminophor and Au@NiFe MOFs as the dualquencher. The as-prepared Au@NiFe MOFs with porous structure could decrease the ECL intensity of Ru(bpy)32+/Zn-oxalate MOFs compound due that both Au nanoparticles and NiFe-based MOFs could quench the ECL efficiency as the ECL acceptor in the RET process, which could significantly improve the sensitivity of the immunosensor. Besides, the dual quenching effect was confirmed by the overlap between the UV-vis absorption spectrum of Au@NiFe MOFs and the ECL emission spectrum of Ru(bpy)32+/Zn-oxalate MOFs compound. Furthermore, the suggested dual quenching ECL immunosensor exhibited outstanding selectivity, stability, and reproducibility, demonstrating a promising quenching strategy to determinate the Aβ.


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It was worth pointing out that the successful use of Ru(bpy)32+/Zn-oxalate MOFs and NiFe-based MOFs in this ECL system also provided the inspiration for other applications of MOFs materials for the researchers in ECL methodology.

ACKNOWLEDGMENTS This study was supported by the Natural Science Foundation of Shandong Province (No. ZR2016BM20), National Natural Science Foundation of China (Nos. 21575050, 21505051, and 21777056), National Key Scientific Instrument and Equipment Development Project of China (No. 21627809), and Qin Wei thanks the Special Foundation for Taishan Scholar Professorship of Shandong Province (No. ts20130937) and UJN.

ASSOCIATED CONTENT Supporting Information Materials and reagents, apparatus, ECL Measurement Procedure, EDS spectrum of Ru(bpy)32+/Zn-oxalate MOFS, FT-IR spectrogram of NiFe-based MOFs and Au@NiFe MOFs, XPS survey spectra of NiFe-based MOFs, stability of the proposed immunosensor, Limit of detection calculation, comparison of the performance of the proposed and referenced immunosensor for Aβ detection, analysis of Aβ in the artificial cerebrospinal.

AUTHOR INFORMATION Corresponding Author *Tel: +86 53182767890. Fax: +86 53187161600. E-mail: [email protected] (W. Cao).



*Tel: +86 53182767890. Fax: +86 53187161600. E-mail: [email protected] (Q. Wei). Notes The authors declare no competing financial interest.


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FIGURE CAPTIONS Scheme 1. The preparation of Ru(bpy)32+/Zn-oxalate MOFs/Ab1 bioconjugate (A) and Ab2-Au@NiFe MOFs bioconjugate (B); construction process of the proposed dual quenching ECL immunosensor (C). Figure 1. SEM image of Ru(bpy)32+/Zn-oxalate MOFs (A); SEM (B) and HRTEM (C) images of NiFe-basded MOFs; SEM (D), HRTEM (E), and EDAX mapping images (F, 1-3) of Au@NiFe MOFs. Figure 2. The PXRD of Ru(bpy)32+/Zn-oxalate MOFs (A); The FT-IR spectrogram of Ru(bpy)32+/Zn-oxalate MOFs (B); The PXRD of NiFe-based MOFs and Au@NiFe MOFs (at the bottom of the graph, the red was the typical diffraction peaks of Fe2O2CO3 with the JCPDS card 33-0665, and the black was the typical diffraction peaks of Fe0.93Ni0.056 with the JCPDS card 44-1088) (C); The EDS spectrum and atomic percentages of Au@NiFe MOFs (D). Figure 3. The XPS spectra of Ni 2p (A), Fe 2p (B), and C 1s (C) for NiFe-based MOFs. Figure 4. (A) UV-vis absorption spectrum of NiFe-based MOFs (a) Au@NiFe MOFs (b), and ECL emission spectrum of Ru(bpy)32+/Zn-oxalate MOFs (c); (B) Fluorescence emission spectrum of Ru(bpy)32+/Zn-oxalate MOFs (a), Ru(bpy)32+/Zn-oxalate MOFs/NiFe-based MOFs (b), and Ru(bpy)32+/Zn-oxalate MOFs/Au@NiFe MOFs (c); (C) ECL behavior of Ru(bpy)32+/Zn-oxalate MOFs (a), Ru(bpy)32+/Zn-oxalate MOFs/NiFe based MOFs (b), and Ru(bpy)32+/Zn-oxalate MOFs/Au@NiFe MOFs (c). The concentration of Ru(bpy)32+/Zn-oxalate MOFs, NiFe-based MOFs, and Au@NiFe MOFs were 0.5 mg mL-1, 0.8 mg mL-1, and 0.8 mg mL-1, respectively.



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Figure 5. The ECL behavior (A) (in PBS pH = 7.4 containing 10 mmol L-1 tripropylamine) and electrochemical impedance spectroscopy (B) (EIS of 2.5 mmol L1

Fe(CN)63-/4- and 0.1 mol L-1 KCl) of bare GCE (a), GCE/Ru(bpy)32+/Zn-oxalate MOFs

(b),

GCE/Ru(bpy)32+/Zn-oxalate

MOFs/Ab1/BSA

(d),

MOFs/Ab1

(c),



GCE/Ru(bpy)32+/Zn-oxalate MOFs/Ab1/BSA/Aβ

MOFs/Ab1/BSA/Aβ/Ab2-Au@NiFe

MOFs(f).

(e), The

concentration of Aβ was 50 ng mL-1. Figure 6. The influence of pH (A), concentration of tripropylamine (B), concentration of Ru(bpy)32+/Zn-oxalate MOFs (C), concentration of Au@NiFe MOFs (D) (the concentration of Aβ was 50 ng mL-1) on ECL response. Error bars = SD (n=3). Figure 7. ECL response (A) and calibration curve (B) of the immunosensor for different concentrations of Aβ (0.0001, 0.0005, 0.005, 0.01, 0.1, 1, 10, 25, and 50 ng mL-1). Reproducibility (C) (concentration of Aβ was selected as 50 ng mL-1) and selectivity (D) (concentration of Aβ and other interferents were 50 ng mL-1 and 500 ng mL-1 respectively) of the ECL immunosensor. Error bars = SD (n=3).


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Scheme 1.



Figure 1.


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Figure 2.



Figure 3.


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Figure 4.



Figure 5.


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Figure 6.



Figure 7.


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For TOC only


Dual Quenching Electrochemiluminescence Strategy Based on 3D

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