MnCO3 as a New Electrochemiluminescence Emitter for Ultrasensitive

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Functional Inorganic Materials and Devices

MnCO3 as New Electrochemiluminescence Emitter for Ultrasensitive Bioanalysis of #-Amyloid1-42 Oligomers Based on Site-directed Immobilization of Antibody Yue Jia, Lei Yang, Ruiqing Feng, Hongmin Ma, Dawei Fan, Tao Yan, Rui Feng, Bin Du, and Qin Wei ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b21928 • Publication Date (Web): 28 Jan 2019 Downloaded from http://pubs.acs.org on January 29, 2019

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MnCO3 as New Electrochemiluminescence Emitter for Ultrasensitive Bioanalysis of β-Amyloid1-42 Oligomers Based on Site-directed Immobilization of Antibody Yue Jia,† Lei Yang,† Ruiqing Feng,† Hongmin Ma,† Dawei Fan,† Tao Yan,‡ Rui Feng,‡ Bin Du, †,‡ ,* and Qin Wei † ,* †

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

School of Water Conservancy and Environment, University of Jinan, Jinan 250022, China

ABSTRACT: In this work, an electrochemiluminescence (ECL) immunosensor was proposed utilizing MnCO3 nanospheres as a novel ECL luminophor and HWRGWVC (HC-7) heptapeptide as an efficient antibody capturer for site-directed immobilization with high affinity. MnCO3 nanospheres that prepared by homogeneous precipitation method exhibited high ECL efficiency, low toxicity, favorable biocompatibility and excellent stability. After the functionalization of poly dimethyl diallyl ammonium chloride (PDDA), the obtained MnCO3/PDDA could combine with gold nanoparticles (Au NPs) via electrostatic interaction (MnCO 3/PDDA/Au). Besides, HC-7 as a small peptide ligand, has demonstrated to bind Fc portion of antibody with high affinity. Since the end of HC-7 is a cysteine, it can connect to MnCO3/PDDA/Au via Au-S bond. Then, the antibody could be effectively captured by HC-7 through specific interaction with a better maintained activity than traditional coupling reaction. For verifying the practicability of the constructed immunosensor, β-Amyloid1-42 oligomers (Aβ) was employed as an analyte. Based on the above points, the immunosensor performed favorable ECL property to Aβ concentrations in a wide linear range (0.1 pg/mL-10 ng/mL) with a low detection limit (19.95 fg/mL). With excellent repeatability, selectivity and stability, this method opened up a new avenue for realizing the ultrasensitive detection of Aβ and other biomarkers in real sample analysis. KEYWORDS: electrochemiluminescence, immunosensor, MnCO3, site-directed antibody immobilization, β-Amyloid1-42 oligomers

1. INTRODUCTION Alzheimer's disease (AD) threatens the life and health of the aged seriously as a neurodegenerative disease that cannot be cured and can only be prevented in medical field.1-3 It has been confirmed that β-Amyloid1-42 oligomers (Aβ) as a kind of human cellular secretion has a strong toxic effect on the brain nerve,4-5 which can kill the local brain tissue, hurt the peripheral neuron and trigger gliosis, causing premature apoptosis, and is a major biomarker of AD. Therefore, it will be of great significance for the early diagnosis and prevention of AD if the rapid detection of Aβ can be achieved in human cerebrospinal fluid (CSF).6 Electrochemiluminescence (ECL) immunosensor has a wide application in modern immunoassay due to its excellent controllability, strong immune recognition and low detection limit.7-8 In recent years, new ECL sensing strategies based on nanomaterials or nanocomposites9-10 has received widespread attention besides widely studied ECL reagents such as Ru(bpy)32+, luminol and quantum dots.11-13 Currently, some semiconductor materials such as Cd-based nanomaterials (CdSe, CdS and CdTe) with size-tunable luminescence and high quantum yield are becoming one of the most promising ECL emitters in the future.1416 However, their applications were limited in biological and immunoassay due to unavoidable biotoxicity.17-18 Therefore, it is an urgent problem to find ECL materials with good biocompatibility based on the nontoxic physiology.

MnCO3, a familiar battery material and industrial catalyst, exhibited high ECL efficiency in the presence of K2S2O8.19-21 Specifically, compared with some heavy metal based semiconductors or quantum dots, MnCO3 with excellent biocompatibility is suitable for application in ECL immunoassay.22-23 However, this topic is rarely studied. Furthermore, for enhancing the sensitivity of immunosensor, some assisted approaches and innovative strategies should be included. The immune-recognition ability of ECL immunosensor depends not only on the amount of antibodies, but on the activity of immobilized antibodies.24-26 Although sensor surface prepared with antibody immobilized in a random manner has obtained satisfactory results, site-directed immobilization of the sensing molecules significantly improves the antibody activity and immunosensor sensitivity. Currently, the common strategy of sitedirected antibody fixation was using protein ligands binding to the Fc portions of antibody via the specific affinity to protect the Fab portions for identifying antigens,27-28 such as protein A and protein G.29-30 However, some peptide ligands with stable structure, popular price and good specific affinity to antibody are expected to be explored due to above protein ligands have the disadvantages of poor specificity and narrow binding range with antibody. HWRGWVC (HC-7)31 effectively solves this issue as an altered heptapeptide, which also possess good recognition and affinity to Fc domains, could be used to replace protein ligands to fabricate the immunosensor, completely.

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Scheme 1. Schematic representation of the fabrication of immunosensor.

In this work, MnCO3 with excellent ECL property was synthesized by controlling the volume ratio of water and glycol and utilized as a new ECL emitter in a immunosensor fabrication for Aβ detection. By the successful function of PDDA, the obtained MnCO3/PDDA can adsorb large amount of Au NPs via electrostatic interaction. In order to maintain antibody activity and improve immunosensor sensitivity, HC-7 heptapeptide with high immune affinity was employed to capture Fc portion of antibody. Finally, a label-free immunosensor was fabricated based on MnCO3/PDDA/Au/HC-7 as an innovative ECL sensing platform for ultrasensitive analysis of the Aβ in human CSF with favorable specificity, stability and repeatability.

2. EXPERIMENTAL METHODS 2.1 Preparation of MnCO3/PDDA/Au. MnCO3 was synthesized as follows.32 First, 0.015 M of MnSO4 in a mixture of water and glycol (50% v/v) were under vigorous stirring until the solid dissolved before 0.15 M of NaHCO3 solution was added. The mixed solution containing MnSO4 and NaHCO3 was stirred for 4 h at 25 oC to get the turbid liquid. The obtained precipitates were filtered and washed before drying at 60 oC for 12 h. After that, 15 mg MnCO3 powder was added 40 mL of mixed solution containing 10 mL of PDDA (2 wt%) and 10 mL of hydrazine hydrate solution (4 wt%), the mixture was kept stirring at 100 oC for 30 min to obtain MnCO3/PDDA.33 Then, 1.5 mg MnCO3/PDDA was added in 5 mL of Au NPs sol and the mixture was oscillated overnight to obtain the compound of MnCO3/PDDA/Au (the specific preparation steps of Au NPs are described in Supporting Information). After centrifugation, the

final product was obtained and dispersed in 1 mL of deionized water for following use. 2.2 Fabrication of ECL immunosensor. The glassy carbon electrode (GCE) with diameter of 4 mm was employed as working electrode and it was washed with water and ethanol by using ultrasound for 30 s, respectively. Then, the electrode was polished by using alumina slurries with different granularity (1.0, 0.3 and 0.05 μm) and blown dry with nitrogen. Next, Scheme 1 clearly exhibited the fabrication process of the immunosensor, as follows. Firstly, 6 μL of MnCO3/PDDA/Au suspensoid was applied to prepared GCE surface and dried. Later, the electrode was immersed in HC-7 solution with 5 μg/mL for 30 min and dried film. Subsequently, 6 μL of BSA (0.1 wt%) were dropped on the dry film for blocking the non-specific active sites. Then, 6 μL of anti-Aβ were incubated on the dry film at 4 oC refrigerator, and removed the excess anti-Aβ by washing with the PBS (pH=7.4). Finally, Aβ with a series of concentrations were incubated on the electrode and maintained at 37 oC for following use. 2.3 RP-HPLC analysis of HC-7. The detection of HC-7 was proceeded by a reverse phase high-performance liquid chromatogr (RP-HPLC) system equipped with a Zorbax C18 column (250×4.6 mm i.d., 5 μm) and a diode array detector. HC-7 were injected into a sample loop of 20 μL by a microsyringe. The sixport valve was switched to the ‘inject’ state, and the targets were eluted into the column for separation with HPLC mobile phase (Acetonitrile-Water, 30:70, v/v). And then the targets were further detected at wavelength of 220 nm with a diode array detector. Additionally, all flow rate of mobile phase was 0.5 mL/min.

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Figure 1. SEM images (A) (B) and XRD pattern (C) of MnCO 3. SEM image (D) and EDS pattern (E) of MnCO 3/PDDA/Au. Zeta potentials (F) of MnCO 3, PDDA, MnCO3/PDDA and Au NPs sol.

Figure 2. (A) RP-HPLC chromatograms of HC-7 in 5 μg/mL concentration and its supernatant coupled with MnCO 3/PDDA/Au. (B) RP-HPLC detection of HC-7 with different concentrations: 10 ng/mL, 50 ng/mL, 100 ng/mL, 500 ng/mL, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL, 20 μg/mL, 30 μg/mL. The effect of HC-7 on the efficiency of immunosensor fabrication including (C) incubation time and (D) antibody concentration. (E) UV -vis absorption detection of anti-Aβ with different concentrations from 0 to 12 μg/mL. (F) CD spectrums of anti-Aβ coupled with different substrates.

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2.4 UV-vis absorption spectra and CD analysis of anti-Aβ. A TU-1909 ultraviolet spectrophotometer system was used to detect the absorbance of anti-Aβ at λmax=276 nm for calculating the amount of antibody binding with different substrates. The circular dichroism (CD) spectra was employed to detect the antiAβ secondary structure for determining their activity. 2.5 ECL measurements of Immunosensor. The immunosensor was scanned by a three-electrode system in a mixed electrolyte containing 1/15 M PBS (pH=7.0), 0.1 mM K2S2O8 and 0.1 mM KCl. Cyclic voltammetry (CV) was used for the electrochemical measurement from -1.8 to 0 V at scanning rate of 150 mV/s. Under optimal conditions, the ECL responses to Aβ with different concentrations were detected.

3. RESULTS AND DISCUSSION 3.1 Characterization of MnCO3 and MnCO3/PDDA/Au. The morphologies of MnCO3 and MnCO3/PDDA/Au were characterized by scanning electron microscope (SEM). In Figure 1A and Figure 1B, the favorable monodispersed MnCO3 nanomaterials exhibited cubic and spherical structure with the diameter about 750 nm. And the particles surface with lots of irregular bulges and folds exhibited large surface area that could adsorb a lot of Au NPs. As expected, after the functionalization of PDDA, the obtained MnCO3/PDDA could combine with Au NPs (Figure 1D). To further prove this conclusion, the energy dispersive spectrometer (EDS) was employed to analyze the elemental composition of MnCO3/PDDA/Au, and the results were shown in Figure 1E, the nanospheres were mainly composed of C, Mn, O and Au elements, which confirmed that the successful combination of Au NPs on MnCO3/PDDA surface. The X-ray diffraction (XRD) was applied to find out the crystallization of MnCO3. Multiple sharp peaks were found at 2θ=24.3o, 31.4o, 37.5o, 41.4o, 45.2o, 51.5o and 51.7o (Figure 1C), which corresponded to the (012), (104), (110), (113), (202), (024) and (116) planes of MnCO3, respectively.32, 34 The results proved the successful preparation of MnCO3. In addition, the Zeta potential was used to analyze the charge characteristic of different materials, providing a theoretical basis for the electrostatic adsorption of MnCO3 and Au NPs. The results were shown in Figure 1F, the Zeta potentials of MnCO3/PDDA and Au NPs were 38.8 mV and -32.9 mV, respectively, thus Au NPs could be adsorbed on MnCO3 surface via electrostatic attraction, further confirmed the successful synthesis of MnCO3/PDDA/Au composites. 3.2 RP-HPLC characterization of HC-7. The coupling of HC-7 heptapeptide with MnCO3/PDDA/Au can be analyzed by RP-HPLC and the detection results were shown in Figure 2A. 5 μg/mL of HC-7 was detected at 2.2 min (curve a), while the peak area was significantly diminished (curve b) after that incubated with MnCO3/PDDA/Au. It proved that the successful incubation of HC-7 with MnCO3/PDDA/Au. Sequentially, to calculate the amount of combinations of HC-7 with MnCO3/PDDA/Au, the RP-HPLC peak area of the linear relationship was obtained by detecting a series of HC-7 concentrations range from 10 ng/mL-30 μg/mL. The linear fitting equation was y= -22.18406+86.19236×lg c with the correlation coefficient of 0.996 (Figure 2B), and it can be calculated that the load capacity of HC-7 with MnCO3/PDDA/Au was 2.35 μg/mg. The experimental results indicated that the vast majority of HC-7 were incubated successfully, and there were no aggregations of ligand-ligand.

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with cysteines-end via Au-S bond, while the other end could specifically capture Fc domains of antibody under lower concentration in shorter time, so as to reduce the uncontrollability of antibody random binding with Au NPs. The reason was that the specific recognition efficiency of HC-7 with Fc domains was much higher than the formation efficiency of Au-N bond, which led to a satisfactory incubation effect despite the decrease of antibody concentration and incubation time. To prove the above views, the absorbance of anti-Aβ with different concentrations was detected at λmax=276 nm.35 As shown in Figure 2E, the absorbance of the linear relationship was obtained by detecting a series of anti-Aβ with different concentrations ranging from 0 to 12 μg/mL at λmax=276 nm. The linear regression equation was A=9.55046×104 +0.00923×c with the correlation coefficient of 0.998. In this way, the amount of antibody incubation can be calculated by Aimmobilized =Afeed - Aunbound, and the feasibility of HC-7 could be analyzed similarly. As shown in Figure 2C, broken line (a) and (b) represented the antibody incubation curve over time in the case of contain HC-7 and without it, respectively. It can be clearly seen that the incubation amount of anti-Aβ rapidly increased from 0 to 30 min and remained almost unchanged more than 30 min (broken line a). However, broken line (b) did not reach saturation until 480 min later, and the total incubation amount were slightly less than broken line (a). Furthermore, Figure 2D showed the broken line of incubation ability of different substances to anti-Aβ with different concentrations at same time. The broken line (c) reflected the incubation situation of MnCO3/PDDA/Au/HC-7. It can be seen that when the anti-Aβ concentration was only 2 μg/ml, the incubation amount can be up to 1.2 μg/mg. But the incubation amount of the substrate without HC-7 (broken line d) was only 0.9 μg/mg when the anti-Aβ concentrations were higher. The above experimental results indicated that the existence of the HC7 heptapeptide shortened the incubation time of anti-Aβ, which confirmed the higher specific-binding efficiency of antibody using HC-7 than Au-N bond method. In addition, HC-7 could significantly reduced the dosage of anti-Aβ based on its strong immune recognition. In addition to strong immune recognition ability, HC-7 can significantly maintain the activity of antibody, and the viewpoint was validated by using CD spectrum to detect the anti-Aβ secondary structure. The results were obtained for the conformational analysis of the anti-Aβ showed in Figure 2F. The curve (a) represented the CD spectrum of solvent and MnCO3/PDDA/Au, which showed no CD absorption. This indicated that the solvent and MnCO3/PDDA/Au will not affect the data of anti-Aβ and these are suitable for the CD detection. Furthermore, curve (b) and (c) represented the CD spectrum of the same amount of anti-Aβ combined with MnCO3/PDDA/Au and MnCO3/PDDA/Au/HC-7, and both showed the pure α-helical conformation due to the characteristic negative peaks appeared at 208 nm and 222 nm.36-37 However, the CD absorption intensity of curve (b) without HC-7 was lower than that of curve (c) with HC7 added, indicating that sample (b) has less α-helical compared to the sample (c). The reason was that the anti-Aβ will bind to Au NPs directly in a random way without HC-7. So that it will inactivate and reduce the number of α-helical37-38 if the binding site at the Fab domains due to its secondary structure have been destructed, which lead to the decrease of the characteristic peak absorption value. On the contrary, the CD spectrum of sample (c) was significantly stronger than that of sample (b) due to the presence of HC-7 which could specifically identify the Fc domains and expose Fab domains to protect the secondary structure of anti-Aβ, thus the survival rate of antibody incubation

3.3 Feasibility analysis of the HC-7 for immunosensor fabrication. The advantage of HC-7 is that can connect Au NPs

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Figure 3. (A) ECL-potential profiles, (B) EIS curves, (C) ECL-time profiles and (D) CV curves of immunosensors with different modified states: (a) GCE, (b) GCE/MnCO3/PDDA/Au, (c) GCE/MnCO3/PDDA/Au/HC-7, (d) GCE/MnCO3/PDDA/Au/HC-7/BSA, (e) GCE/MnCO3/PDDA/Au/HC-7/BSA/anti-Aβ, (f) GCE/MnCO3/PDDA/Au/HC-7/BSA/anti-Aβ/Aβ.

Figure 4. (A) ECL responses of the immunosensor towards Aβ concentrations from 0.1 pg/mL to 10 ng/mL. (B) Linear fitting curve for the Aβ detection.

improved a lot. The above experimental results showed that the strategy of antibody site-directed immobilization using HC-7 heptapeptide improved the survival rate of antibody and immunosensor recognition compared to the traditional random binding, as expected. Therefore, HC-7 was feasible for immunosensor fabrication, completely. 3.4 Mechanism investigation of MnCO3-K2S2O8 ECL system. Discussion on the ECL mechanism of MnCO3-K2S2O8 system was based on relevant references reports.10, 21 Figure S3 showed the CV scanning curve of MnCO3 in K2S2O8. solution. When the modified electrode was scanned from 0 ~ -0.6 V, the reaction (1) was the main process. During this period, S2O82•— captured electrons to produce SO4 radical with strong oxidizing property. As the potential became more negative, the electrode reaction (2) began to occur, which is carried out simultaneously with electrode reaction (1), the increased current suddenly could •— •— verify it. Subsequently, MnCO3 was oxidized by SO4 and excited to a high energy state MnCO3* (reaction 3). During the transition from the excited state to the ground state, the stronger ECL signal was released (reaction 4). –

S2O82 + e → SO4

•—

+ SO42•—

MnCO3 + e → MnCO3 SO4

•—

•—

+ MnCO3 *

→ MnCO3* + SO42-

MnCO3 → MnCO3 + hv

(1) (2) (3) (4)

3.5 Electrochemistry characterization of the immunosensor fabrication. Electrochemical characterization is an important method to support the stepwise fabrication of immunosensor. Figure 3A and 3C showed the ECL-potential curves and ECLtime profiles of the electrode under different modified states, which were carried out in PBS (pH=7.0) containing 0.1 M K2S2O8 and 0.1 M KCl. A weak ECL signal was produced on bare electrode (curve a). When the composite of MnCO3/PDDA/Au was modified on the surface of GCE, the ECL signals increased greatly (curve b), it can be explained that the Au NPs effectively increased the electron transfer rate on the MnCO3/PDDA surface and accelerated ECL emission. Subsequently, as HC-7, BSA, anti-Aβ and Aβ were immobilized on the surface of the electrode, the ECL intensity showed a decreasing trend (curve c, d, e and f), which could be explained by the non-conductivity of protein and peptides that hinder the electron transfer. The above experimental results confirmed the immunosensor was constructed successfully. EIS is one of the most commonly used electrochemical methods to characterize the assembly process of immunosensor, which could clarify the interface properties of the electrodes and further improve the reliability of ECL-potential results. Figure 3B exhibited the original curves of EIS with different modified electrodes, which are carried out in a electrolyte containing 2.5 mM [Fe(CN)6]3-/4- and 0.1 M KCl. The curve (a) and (b) represented that the EIS of bare GCE and MnCO3/PDDA/Au modified electrode, respectively, which both exhibited a low impedance value because of the favorable electrical conductivity of bare GCE and MnCO3/PDDA/Au. With the sequential

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modification of HC-7, BSA, anti-Aβ and Aβ, the semicircle domains showed a rising tendency (curve c, d, e and f), indicating the higher electron transfer resistance. The experimental results further proved the successful fabrication of the immunosensor. CV curves also could support the stepwise fabrication of immunosensor. Figure 3D showed the CV date of the electrode with different modification states in electrolyte containing 2.5 mM [Fe(CN)6]3- and 0.1 M KCl. It can be seen that the bare GCE has the largest peak current. But as MnCO3/PDDA/Au, HC-7, BSA, anti-Aβ and Aβ with poor electrical conductivity were modified on the electrode surface, the peak current presented a decreasing trend. This indicated that the above substances can be successively bonded to the electrode surface. From what has been discussed above, the proposed immunosensor was fabricated successfully. 3.6 Analysis of Aβ. In this work, Aβ with different concentrations were detected to analyze the performance of the immunosensor under the optimal conditions (the optimization analysis of experimental conditions are shown in supporting information). Figure 4A exhibited the trend in ECL intensity by detecting a series of Aβ with concentrations from 0.1 pg/mL-10 ng/mL. The calibration curve (Figure 4B) was displayed by plotting the logarithm of Aβ concentration against the ECL intensity with the equation was IECL = 3296.49 - 726.26×lg c (R=0.997). By calculating, the ultralow detection limit was obtained of 19.95 fg/mL (S/N = 3), which is far lower than that of other detection methods (Table S1). The above experimental results proved that the immunosensor with great sensitivity could achieve the detection of Aβ at femtogram level based on the sitedirected antibody immobilization, as expected. 3.7 Stability, selectivity, and repeatability of the ECL immunosensor. Stability, selectivity and repeatability are three of the most important indexes to judge the performances of immunosensor. In view of this, these three properties were shown in Figure S2 with multiple detection results. It could be clearly seen from the figure that the immunosensor has good stability, selectivity and repeatability with all the relative standard deviation (RSD) were less than 1.36%. 3.8 Application in human CSF sample. Real sample analysis is vital in immunosensor for target detection.39-40 In this work, the standard addition method was employed to detect the feasibility and veracity of immunosensor. 41-42 The results were obtained by detecting different concentration of Aβ in human CSF sample. Subsequently, the ECL signal was detected by adding three standard concentrations of Aβ (1.00, 3.00, 5.00 ng/mL) into original samples and the detection date was entered in Table S2 with the RSD of Aβ in human CSF samples were less than 6.45% and the recoveries43 were from 98% to 102%, proving that the immunosensor could apply to the analysis of Aβ sensitively.

4. CONSLUSIONS In conclusion, an ultrasensitive ECL immunosensor was designed and successfully applied to detect the Aβ in real samples. MnCO3 were successfully synthesized and used as new luminophore in an enhanced ECL immunosensing system. Besides, HC-7 heptapeptide was utilized to realize the sitedirected immobilization of antibody, it shortened the incubation time with a better kept antibody activity, which improved the sensitivity remarkably. Also, the immunosensor performed favorable ECL property to Aβ with the concentrations from 0.1 pg/mL-10 ng/mL and the ultralow detection limit of 19.95 fg/mL. It is believed that this sensing strategy will provide new prospective for realizing the sensitive detection of Aβ and other biomarkers in real samples.

■ ASSOCIATED CONTENT

Supporting Information Materials and reagents; apparatus; preparation of Au NPs; optimization of experimental conditions (Figure S1); stability, selectivity, and repeatability of the ECL immunosensor (Figure S2); the CV curves of MnCO3 in K2S2O8 solution (Figure S3); the actual application of the immunosensor detection data (Table S1); comparison of the results of different detection methods of Aβ (Table S2).

■ AUTHOR INFORMATION Corresponding Author *

E-mail: [email protected] (Bin Du)

Tel: +86 531 82767872 Fax: +86 531 82767367

*E-mail: [email protected] (Qin Wei) Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This work was supported by the National Key Scientific Instrument and Equipment Development Project of China (No. 21627809), and the National Natural Science Foundation of China (Nos. 21575050, 21777056, 21505051).

■ REFERENCES (1)

Blennow, K.; Leon, M. J. D.; Zetterberg, H. Alzheimer's Disease. Lancet. 2006, 368, 387-403. (2) Ballard, C.; Gauthier, S.; Corbett, A.; Brayne, C.; Aarsland, D.; Jones, E. Alzheimer's Disease. Lancet. 2011, 377, 1019-1031. (3) Qu, F.; Yang, M.; Rasooly, A. Dual Signal Amplification Electrochemical Biosensor for Monitoring the Activity and Inhibition of the Alzheimer's Related Protease β-Secretase. Anal. Chem. 2016, 88,10559-10565. (4) Carneiro, P.; Loureiro, J.; Delerue-Matos, C.; Morais, S.; Pereira, M. D. C. Alzheimer’s Disease: Development of a Sensitive Label-Free Electrochemical Immunosensor for Detection of Amyloid beta Peptide. Sens. Actuators. B. Chem. 2017, 239, 157-165. (5) Kim, S. J.; Seo, Y.; Kim, H. J.; Na, D. L.; Seo, S. W.; Kim, Y.; Suh, Y. L. Pathologically Confirmed Cerebral Amyloid Angiopathy with No Radiological Sign in a Patient with Early Onset Alzheimer's Disease. Yonsei. Med. J. 2018, 59, 801-805. (6) Selkoe, D. J. Alzheimer's Disease: Genes, Proteins, and Therapy. Physiol. Rev. 2001, 81, 741-766. (7) Li, L.; Chen, Y.; Zhu, J. J. Recent Advances in Electrochemiluminescence Analysis. Anal. Chem. 2016, 89, 358-371. (8) Knight, A. W.; Greenway, G. M. Relationship between Structural Attributes and Observed Electrogenerated Chemiluminescence (ECL) Activity of Tertiary Amines as Potential Analytes for the Tris (2,2'-Bipyridine) Ruthenium(II) ECL Reaction. Analyst. 1996, 121, 101R-106R. (9) Yang, L.; Zhu, W.; Ren, X.; Khan, M. S.; Zhang, Y.; Du, B.; Wei, Q. Macroporous Graphene Capped Fe3O4 for Amplified Eectrochemiluminescence Immunosensing of Carcinoembryonic Antigen Detection Based on CeO2@TiO2. Biosens. Bioelectron. 2017, 91, 842-848. (10) Lei, Y.; Zhou, J.; Chai, Y.; Zhuo, Y.; Yuan, R. SnS2 Quantum Dots as New Emitters with Strong Electrochemiluminescence for Ultrasensitive Antibody Detection. Anal. Chem. 2018, 90, 12270-12277. (11) Yang, L.; Li, Y.; Zhang, Y.; Fan, D.; Pang, X.; Wei, Q.; Du, B. 3D Nanostructured Palladium Functionalized Graphene Aerogel Supported Fe3O4 for Enhanced Ru(bpy)32+-based

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Electrochemiluminescent Immunosensing of Prostate Specific Antigen. ACS. Appl. Mater. Interfaces. 2017, 9, 35260-35267. Xing, B.; Zhu, W.; Zheng, X.; Zhu, Y.; Qin, W.; Dan, W. Electrochemiluminescence Immunosensor Based on Quenching Effect of SiO2 @PDA on SnO2/rGO/Au NPs-Luminol for Insulin Detection. Sens. Actuators. B. Chem. 2018, 265, 403411. Ma, H.; Zhao, Y.; Liu, Y.; Zhang, Y.; Wu, D.; Li, H.; Wei, Q. A Compatible Sensitivity Enhancement Strategy for Electrochemiluminescence Immunosensors Based on the Biomimetic Melanin-Like Deposition. Anal. Chem. 2017, 89, 13049-13053. Rogach, A. L.; Franzl, T.; Klar, T. A.; Feldmann, J.; Gaponik, N.; Lesnyak, V.; Shavel, A.; Eychmüller, A.; And, Y. P. R.; Donegan, J. F. Aqueous Synthesis of Thiol-Capped CdTe Nanocrystals:  State-of-the-Art. J. Phys. Chem. C. 2007, 40, 14628-14637. Peng, X.; Manna, L.; Yang, W.; Wickham, J.; Scher, E.; Kadavanich, A.; Alivisatos, A. P. Shape Control of CdSe Nanocrystals. Nature. 2000, 404, 59-61. Lv, X.; Pang, X.; Li Y.; Yan, T.; Cao, W.; Du, B.; Wei, Q. Electrochemiluminescent Immune-Modified Electrodes Based on Ag2Se@CdSe Nanoneedles Loaded with Polypyrrole Intercalated Graphene for Detection of CA72-4. ACS. Appl. Mater. Interfaces. 2015, 7, 867-872. Sharifi, S.; Behzadi, S.; Laurent, S.; Forrest, M. L.; Stroeve, P.; Mahmoudi, M. Toxicity of Nanomaterials. Chem. Soc. Rev. 2012, 41, 2323-2343. Ma, C.; Wu, W.; Peng, Y.; Wang, M. X.; Chen, G.; Chen, Z.; Zhu, J. J. A Spectral Shift-Based Electrochemiluminescence Sensor for Hydrogen Sulphide. Anal. Chem. 2017, 90, 13341339. Lei, W.; Sun, Y.; Zeng, S.; Cui, C.; Li, H.; Xu, S.; Wang, H. Study on the Morphology-Controlled Synthesis of MnCO3 Materials and their Enhanced Electrochemical Performance for Lithium Ion Batteries. CrystEngComm. 2016, 18, 8072-8079. Gang, W.; Huang, B.; Lou, Z.; Wang, Z.; Qin, X.; Zhang, X.; Ying, D. Valence State Heterojunction Mn3O4/MnCO3: Photo and Thermal Synergistic catalyst. Appl. Catal. B. 2016, 180, 612. Sun, X.; Li Z.; Cheng Y.; Xue L. Study for the Preparation of Shuttle-Like MnCO3 Nanostructures and their Electrochemiluminescence Properties. ECS. J. Solid. State. Sc. 2017, 6, R63-R67. Liu, B.; Zheng, Y. F. Effects of Alloying Elements (Mn, Co, Al, W, Sn, B, C and S) on Biodegradability and in Vitro Biocompatibility of Pure iron. Acta. Biomater. 2011, 7, 14071420. He, Z.; Xiao, Y.; Zhang, J. R.; Zhang, P.; Zhu, J. J. In Situ Formation of Large Pore Silica-MnO2 Nanocomposites with H+/H2O2 Sensitivity for O2-Elevated Photodynamic Therapy and Potential MR Imaging. Chem. Commun. 2018, 54, 29612965. Elena, J. F.; Antonio, C.; Pedrajas, J. R.; Lechuga, L. M. SiteDirected Antibody Immobilization Using a Protein A-Gold Binding Domain Fusion Protein for Enhanced SPR Immunosensing. Analyst. 2013, 138, 2023-2031. Noah, N. M.; Omole, M.; Stern, S.; Zhang, S.; Sadik, O. A.; Hess, E. H.; Martinovic, J.; Baker, P. G. L.; Iwuoha, E. I. Conducting Polyamic Acid Membranes for Sensing and SiteDirected Immobilization of Proteins. Anal. Biochem. 2012, 428, 54-63. Yakovleva, J.; Davidsson, R.; Lobanova, A.; Bengtsson, M.; Eremin, S.; Laurell, T.; Emnéus, J. Microfluidic Enzyme Immunoassay Using Silicon Microchip with Immobilized Antibodies and Chemiluminescence Detection. Anal. Chem. 2002, 74, 2994-3004. Hong Yan, S.; Xiaodong, Z.; Jonathan, H.; Xiaodi, S. Comparative Study of Random and Oriented Antibody Immobilization as Measured by Dual Polarization Interferometry and Surface Plasmon Resonance Spectroscopy. Langmuir. 2012, 28, 997-1004.

(28) Cho, I. H. Site-Directed Immobilization of Antibody Onto Solid Surfaces for the Construction of Immunochip. Biotechnol. Bioproc. E. 2004, 9, 112-117 (29) Ey, P. L.; Prowse, S. J.; Jenkin, C. R. Isolation of Pure IgG1 , IgG2a and IgG2b Immunoglobulins from Mouse Serum Using Protein A-Sepharose. Immunochemistry, 1978, 15, 429-436. (30) Jiang, L.; Dai, Y.; Liu, X.; Wang, C.; Wang, A.; Chen, Z.; Heidbreder, C. E.; Kolokythas, A.; Zhou, X. Identification and Experimental Validation of G Protein Alpha Inhibiting Activity Polypeptide 2 (GNAI2) as a MicroRNA-138 Target in Tongue Squamous Cell Carcinoma. Hum. Genet. 2011, 129, 189-197. (31) Dostalova, S.; Cerna, T.; Hynek, D.; Koudelkova, Z.; Vaculovic, T.; Kopel, P.; Hrabeta, J.; Heger, Z.; Vaculovicova, M.; Eckschlager, T. Site-Directed Conjugation of Antibodies to Apoferritin Nanocarrier for Targeted Drug Delivery to Prostate Cancer Cells. ACS. Appl. Mater. Interfaces. 2016, 8, 1443014441. (32) Yan, Y.; Zhu, Y.; Yu, Y.; Li, J.; Mei, T.; Ju, Z.; Qian, Y. MnCO3 Microstructures Assembled with Nanoparticles: ShapeControlled Synthesis and Their Application for Li-Ion Batteries. J. Nanosci. Nanotechnol. 2012, 12, 7334-7338. (33) Zhou, L.; Kong, X.; Min, G.; Fang, L.; Li, B.; Zhou, Z.; Cao, H. Hydrothermal Fabrication of MnCO3@rGO Composite as an Anode Material for High-Performance Lithium Ion Batteries. Inorg. Chem. 2014, 53, 9228-9234. (34) Shen, X.; Ji, Z.; Miao, H.; Yang, J.; Chen, K. Hydrothermal Synthesis of MnCO3 Nanorods and their Thermal Transformation Into Mn2O3 and Mn3O4 Nanorods with Single Crystalline Structure. J. Alloys. Compd. 2011, 509, 5672-5676. (35) Whitaker, J. R.; Granum, P. E. An Absolute Method for Protein Determination Based on Difference in Absorbance at 235 and 280 nm. Anal. Biochem. 1980, 109, 156-159. (36) NJ, G. Using Circular Dichroism Spectra to Estimate Protein Secondary Structure. Nat. Protoc. 2006, 1, 2876-2890. (37) Buxbaum, E. Protein Secondary Structure. Chem. Biol. Drug. Des. 2010, 19, 394-401. (38) Whitmore, L.; Wallace, B. A. Protein Secondary Structure Analyses from Circular Dichroism Spectroscopy: Methods and Reference Databases. Biopolymers. 2010, 89, 392-400. (39) Ren, X.; Wu, D.; Ge, R.; Sun, X.; Ma, H.; Yan, T.; Zhang, Y.; Du, B.; Wei, Q.; Chen, L. Self-supported CoMoS4 Nanosheet Array as an Efficient Catalyst for Hydrogen Evolution reaction at Neutral pH. Nano. Research. 2018, 11, 2024-2033. (40) Wu, D.; Wei, Y.; Ren, X.; Ji, X.; Liu, Y.; Guo, X.; Liu, Z.; Asiri, A. M.; Wei, Q.; Sun, X. Co(OH)2 NanoparticleEncapsulating Conductive Nanowires Array: RoomTemperature Electrochemical Preparation for HighPerformance Water Oxidation Electrocatalysis. Advanced. Materials. 2018, 30, 1705366-1705372. (41) Ren, X.; Ma, H.; Zhang, T.; Zhang, Y.; Yan, T.; Du, B.; Wei, Q. Sulfur-Doped Graphene-Based Immunological Biosensing Platform for Multianalysis of Cancer Biomarkers. ACS. Appl. Mater. Interfaces. 2017, 9, 37637-37644. (42) Bo, E. H. S.; Kowalski, B. R. Generalized Standard Addition Method. Anal. Chem. 1979, 51, 1031-1038. (43) Hu, Y.; Xu, W.; Li, J.; Li, L. Determination of 5Hydroxytryptamine in Serum by Electrochemiluminescence Detection with the Aid of Capillary Electrophoresis. Luminescence. 2012, 27, 63-68.

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