Dual Signal Amplification Electrochemical Biosensor for Monitoring

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Dual signal amplification electrochemical biosensor for monitoring the activity and inhibition of the Alzheimer related protease #-secretase (BACE1) Fengli Qu, Minghui Yang, and Avraham Rasooly Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b02659 • Publication Date (Web): 21 Sep 2016 Downloaded from http://pubs.acs.org on September 22, 2016

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Analytical Chemistry

Dual signal amplification electrochemical biosensor for monitoring the activity and inhibition of the Alzheimer related protease β-secretase (BACE1)

Fengli Qu†‡, Minghui Yang†*, Avraham Rasooly§*

†

College of Chemistry and Chemical Engineering, Central South University, Changsha,

410083, China ‡

College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu

Shandong, 273165, China § National Cancer Institute, National Institutes of Health, Rockville, MD 20850, USA * Corresponding author: Email: [email protected] (M. Yang); [email protected] (A. Rasooly); Tel: (+86) 731 88836356

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ACS Paragon Plus Environment


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ABSTRACT The protease BACE1 (the β-site amyloid precursor protein cleaving enzyme 1) catalyzes the first step in the synthesis of β-amyloids (Aβ), peptides that accumulate in the brain in Alzheimer’s disease (AD). Measurement of BACE1 activity is important for the development of BACE1 inhibitors to slow or stop AD. To measure BACE1 cleavage of the electrode immobilized substrate peptide, we developed a redox-generating hydroxyapatite (HAP) probe which generates electrochemical current by reaction of the nanoparticle with molybdate (MoO42-). The probe combines alkaline phosphatase (ALP) for dual signal amplification and Aβ antibody to bind the probe to the immobilized peptide substrate on the surface of the electrode. We measured the activity of BACE1 at concentrations ranging from 0.25 to 100 U/mL. The use of the dual signal HAP-ALP probe increased the signal by an order of magnitude compared to HAP-only probe, enabling detection limits as low as 0.1 U/mL. To measure the inhibition of BACE1 activity, the BACE1 inhibitor OM99-2 was added to 25 U/mL of BACE1 in concentrations ranging from 5-150 nM. The observed detection limit of inhibition is 10 nM of OM99-2. These results demonstrate the capabilities of this novel biosensor to measure BACE1 activity and inhibitors of BACE1 activity. To the best of our knowledge this is the first report that reaction of HAP nanoparticles with molybdate can generate electrochemical current. This dual signal amplification strategy can be extended to other electrochemical assays and adapted for a wide array of applications.

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INTRODUCTION Alzheimer's disease (AD) is a slow neurodegenerative disease of the brain that is characterized by symptoms like memory impairment and decline in language, thinking and reasoning skills.1-3 AD is accounts for 60~70% of cases of dementia in the United States and most other countries in the world. The possibility of suffering from AD increases significantly after the age of 70, and it may affect around 50% of persons over the age of 85.4 Although the cause of AD disease is not fully understood, the accumulation of the 40- to 42-residue β-amyloid peptide (Aβ) in the brain is believed to play a major role.5-8 The sequential proteolysis of the transmembrane amyloid precursor protein (APP) by β-secretase [i.e., β-site APP cleaving enzyme 1 (BACE1)] and γ-secretase leads to the production of Aβ peptides. β-secretase initially cleave APP in its luminal domain to produce a membrane bound C-terminal fragment of APP, which is then hydrolyzed by γ-secretase to form Aβ.9,10 The activity of β-secretases is important for Aβ production in vivo11 and it was suggested that BACE1 is the major β-secretase for generation of Aβ peptides by neurons.12 In human studies, increased BACE1 expression is observed in patients with sporadic AD13-16 and the link between BACE1 levels, Aβ load, and AD pathology has been established. The Aβ load is correlated with increased β-secretases activity in sporadic Alzheimer's disease patients.17 These finding indicate that increased BACE1 expression is an important risk factor for sporadic AD. Moreover, it has been shown that BACE1 in the retina has potential as a sensitive biomarker for monitoring early pathological changes in Alzheimer's disease,18 and measuring its levels and activity have been proposed as surrogate biomarkers for AD.19 3



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Animal studies suggest that BACE1 -/- mice deficient in BACE1 are healthy, fertile and appear normal in gross anatomy, tissue histology, hematology and clinical chemistry. These BACE1-/- mice hemizygous for the APP transgene are lacking brain β-amyloid (Aβ) and β-secretase-cleaved APP C-terminal fragments (CTFs), suggesting that BACE1 is the major β-secretase in vivo, and that therapeutic inhibition of BACE1 for the treatment of Alzheimer's disease may be free of mechanism-based toxicity.20 Suppressing the activity of β-secretase has been considered as a possible means of reducing the level of Aβ peptide in the brain, and thus of treating AD.21-23 Several BACE1 inhibitor drugs are in clinical trials for Alzheimer’s disease,24 suggesting that measuring BACE1 activity might be useful for inhibitor development and for early detection of AD. Different methods have been reported for measuring BACE1 levels in human tissues,19 and for screening BACE1 inhibitors. These include enzyme-linked immunosorbent assay (ELISA), fluorescence, and surface plasmon resonance (SPR).25-27 For instance, Zhou and co-workers reported sensitive and continuous screening of inhibitors of BACE1 using a single SPR chip. This method enabled multiple assays to be continuously performed, thereby enhancing sample throughput and reducing assay cost.28 Our group also reported the detection of BACE1 activity using peptide template gold nanoclusters.29 The methodology was based on specific peptide cleavage by BACE1, and the fluorescence intensity of the synthesized gold nanocluster was inversely related to the length of the peptide template. By comparison, electrochemistry is a powerful analytical technique that has the advantages of simple instrumentation, low cost and high sensitivity. Electrochemistry is also one of the well established techniques that have found wide applications in clinical, environmental and industrial arenas.30-32 4


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In this work, we developed an electrochemical sensor to measure BACE1 activity based on a dual signal amplification strategy which utilizes a novel hydroxyapatite (HAP) nanoparticle to bind the peptide substrate of BACE1. Hydroxyapatite (HAP) is a natural mineral form of calcium apatite with the formula of Ca10(PO4)6(OH)2 that exhibits good biocompatibility.33-35 HAP nanoparticles have been widely applied in different areas, including drug delivery, molecular imaging, tissue engineering and biosensing.34,36-38. Compared with metal nanoparticles, hydroxyapatite (HAP) has several advantages such as low-cost, relatively simple synthesis and easy functionalization with biomolecules. In this work molybdate was used as an electrical mediator for signal amplification. The second signal was generated by integrating alkaline phosphatase (ALP) onto HAP nanoparticles. Using this HAP-ALP sensor we studied the inhibition effect of potential inhibitors on the activity of BACE1.

EXPERIMENTAL SECTION Materials and apparatus Alkaline phosphatase (ALP), sodium pyrophosphate decahydrate (Na4P2O7.2H20, PPi), β‑site amyloid precursor protein cleaving enzyme 1 (BACE1) and polyethylenimine (PEI) were obtained from Sigma-Aldrich. The antibody (clone 6E10) that is specific to the EFRHDS segment of Aβ peptides was purchased from Covance Inc. (MA, USA). Hydroxyapatite (HAP) nanoparticles were bought from Aladdin Co. Ltd. (Shanghai, China). A peptide with the sequence CKTEEISEVNLDAEFRHDSGY was synthesized and purified by GL Biochem Co. Ltd. (Shanghai, China). BACE1 inhibitor OM99-2 was purchased from Bachem (Bubendorf, Switzerland). 10 mM Tris-HCl buffer (pH=7.4, 100 mM NaCl ) was used as the buffer solution. Other reagents 5



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were of analytical grade and used without further purification. All stock solutions were prepared with double-distilled water. Electrochemical measurements were performed on a CHI-650D electrochemical workstation (Shanghai CH Instruments Co., China). A conventional three-electrode system was used with a gold electrode (Au, 2 mm in diameter) as the working electrode, an Ag/AgCl electrode as the reference electrode and a platinum wire as the auxiliary electrode. Transmission electron microscopic characterization was carried out on a Titan G2 60-300 transmission electron microscope (TEM, FEI, USA). Preparation of the electrochemical sensor. Before preparation of the electrochemical sensor, a HAP based signal probe was synthesized. To conjugate ALP and Aβ antibody onto HAP, HAP nanoparticles were first dispersed into 1% (m/m) PEI solution for 1 h to introduce amino groups. After centrifugation and extensive washing with buffer solution, the HAP was dispersed into 0.25% glutaraldehyde to react for another 30 min. With another round of centrifugation, the HAP nanoparticles were mixed with solution containing 1 µg/mL of Aβ antibody and 10 µg/mL of ALP for 1 h. The final probe suspension (HAP-ALP, 1 mg/mL) was stored at 4 oC before use. For the preparation of the sensor, 5 µL of 4 µM peptide solution was placed on the gold electrode for 12 h to immobilize the peptide onto the gold electrode surface. After extensive washing, the electrode was treated with 1 mM 6-mercaptohexanol (MCH) for 0.5 h to block the electrode from non specific adsorption. Then, BACE1 solutions of different concentrations were added onto the electrode and incubated at 37 oC for 1 h. After a second wash, the synthesized probe solution was dripped onto the electrode and incubated for another 1 h. After a final wash the electrode was ready for measurement. 6


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To test the inhibition of BACE1, different concentrations of inhibitor were incubated with 25 U/mL of BACE1 for 1 h. Then, the activity of BACE1 was measured. Electrochemical Characterization of the Sensor. To characterize the electrodes, 5 µL of 100 µM PPi solution was dripped onto the modified electrode for 30 min followed by the addition of Na2MoO4 solution (6 mM ) for 20 min. The electrode was then measured in 0.5 M H2SO4.

RESULTS AND DISCUSSION Design of a dual signal amplification HAP-ALP electrochemical biosensor for measuring the activity and inhibition of BACE1.

Fig. 1 depicts the sensing

mechanism for testing activity and inhibition of BACE1. The peptide sequence CKTEEISEVNLDAEFRHDSGY was designed according to a previous study, and can be cleaved by BACE1 into two segments of CKTEEISEVNL and DAEFRHDSGY.28 The length of the peptide would not affect the cleavage as long as the KTEEISEVNLDAEF sequence is included.39 The peptide can be linked to a gold electrode surface due to the presence of a cysteine residue. When the peptide modified electrode was incubated with BACE1, the Aβ antibody-recognition segment (EFRHDS) was cleaved and detached from the electrode surface leading to decreased capture of signal probes onto the electrode.28

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Fig. 1. Schematic illustration showing the dual signal amplification sensing principle of BACE1 activity detection.

Previous report and our own work demonstrated that phosphate ions react with molybdate to form redox molybdophosphate precipitates on the electrode surface, thus generating electrochemical current.40,41 We integrated this with a novel HAP nanoparticle electrochemical sensor utilizing molybdate as electrical mediator. Molybdate was used for generating electrochemical current by the reaction with phosphate groups to form redox molybdophosphate precipitates on the electrode surface. A dual signal amplification strategy combines HAP with ALP to produce HAP-ALP probe. HAP contains significant amounts of phosphate groups, while ALP can also hydrolyze pyrophosphate (PPi) to produce phosphate ions (Pi). The reaction of these phosphate groups with molybdate produces molybdophosphate precipitate on the electrode, 8


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generating electrochemical current.40 To prepare the electrochemical sensor, BACE1 specific and cleavable peptides were linked to a gold electrode surface via thiol groups of the terminal cysteine residues. The Aβ antibody binding the fragment of peptide substrate was conjugated onto the HAP nanoparticle surface to prepare the signal probe. When the sensor was incubated with solution containing active BACE1, the peptide was cleaved, releasing the peptide fragment that can bind to Aβ antibody from electrode surface, resulted in the decreased binding sites for the HAP-ALP probe. So the current intensity of the electrodes which was proportional to the amount of HAP-ALP probe captured, and to the activity of BACE1 detected. Using this method, the inhibition effect of potential inhibitors on the activity of BACE1 was also measured.

Characterization of HAP-ALP nanoparticles TEM images indicated the HAP nanoparticles display rod-like shapes with a length of ~100 nm (Fig. 2A). The size of the nanoparticles was also proved by dynamic light scattering (DLS) measurement (Fig. 2B). To test the signal amplification strategy of the HAP based probe, the reaction of HAP with molybdate was first studied. The 1 mg/mL HAP nanoparticle solution was reacted with 6 mM sodium molybdate on the electrode for 20 min and tested in 0.5 M H2SO4. The cyclic voltammograms (CVs) of the resulting electrode displayed two pairs of redox peaks at around 0.22 V and 0.37 V, which supports the formation of molybdophosphate (PMo12O40) precipitate on the electrode surface.40,41 The peaks are due to electron-transfer between different redox states of Molybdate in the precipitate (Fig 3A, curve a). No obvious current peaks were detected in control experiments with only sodium molybdate (Fig 3A curve b). 9



A

60

Intensity (%)

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40

20

0 10

100

1000

Size, nm

B Fig. 2 (A) TEM image of the HAP nanoparticles; (B) Size distribution of the HAP nanoparticles The signal amplification of ALP was studied as follows. PPi was selected as substrate for ALP since ALP can hydrolyze PPi into 2 equivalents of Pi. The resulting Pi then reacts with molybdate to generate electrochemical current. For the substrate PPi, the reaction of 100 µM of PPi with 6 mM sodium molybdate solution also resulted in two pairs of rather weak redox peaks (Fig 3B, curve a). However, after incubation of PPi with ALP for 1 h the redox current of the electrode was increased significantly (Fig 3B, curve b). With the conjugation of Aβ antibody and ALP onto HAP through the amino groups of PEI, the electrochemical performance of the nanoprobe was also studied. The reaction of 10


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the nanoprobe first with PPi and then with molybdate leads to an electrochemical current higher than that of HAP nanoparticles alone (Fig 3C), indicating that ALP conjugated onto HAP participated in the electrochemical reaction. The above experiments successfully demonstrate the dual signal amplification of the HAP-ALP based probe. The corresponding equation regarding the reaction of phosphate with molybdate (equation 1) and redox reaction of molybdophosphate (equation 2 and 3) are displayed below:40,41 12MoO42- + 24 H+ + PO43- = PMo12O403- + 12 H2O

(1)

PMo12O403- + 2e + 2H+ = H2PMo2V Mo10VI O403-

(2)

H2PMo2V Mo10VI O403- + 2e + 2H+ = H4PMo4V Mo8VI O403- (3)

Fig. 3. (A) CV of electrode after reaction of HAP nanoparticle with molybdate (a) and (b) 11



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control experiment with only sodium molybdate resulted in no current peaks (B) CV of electrode after the reaction of 100 µM PPi with 6 mM sodium molybdate solution (curve a), CV of electrode after PPi was hydrolyzed by ALP and then reacted with 6 mM sodium molybdate (curve b). (C) electrochemical performance of Aβ antibody and ALP modified HAP, CV of electrode after reaction of the nanoprobe with molybdate.

Electrochemical characterization of the Sensor. After the immobilization of the cysteine-terminated BACE1 substrate onto the electrode, the electrode was characterized by CV in [Fe(CN)6]3/4 solution. The peak current of the peptide-modified electrode was completely diminished (Fig 4A curve b) compared to that of the bare gold electrode (Fig 4A curve a), confirming the formation of an insulating monolayer on the electrode surface. After the electrode was treated with MCH to block the residual active sites, they were incubated with the HAP-ALP probe solution. The probe could recognize the EFRHDS peptide fragment and be captured on the electrode surface. At this time, with the addition of PPi and then molybdate onto the electrode, the square wave voltammograms (SWVs) of the electrode display two strong redox peaks (Fig 4B, curve a). A previous study reported efficient electron transfer across an insulating monolayer modified electrode with the adsorption of conductive materials onto the insulating monolayer.42-44 The redox current was considered as background current. However, when the peptide modified electrode was treated with 2.5 U/mL of BACE1 solution, the peptide fragment that can bind to Aβ antibody was cleaved and diffused away from the electrode surface. The redox current was correspondingly decreased (Fig 4B, curve b) demonstrating the utility of the electrochemical sensor for assaying BACE 1 activity. 12


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a

-60

0

A

-40

B

-2

-20

Current, µ A

Current, µ A

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b

0 20

-4

b -6

40 -8

60 -0.3

0.0

0.3 E vs Ag/AgCl (V)

0.6

-10 0.1

a 0.2

0.3 0.4 E vs Ag/AgCl (V)

Fig. 4. (A) CV of blank (curve a) and peptide modified (curve b) electrode in [Fe(CN)6]3/4 solution. (B) SWV of the peptide modified electrode without (a) and with the treatment of 2.5 U/mL of BACE1.

Analytical performance of the dual signal sensor. After demonstrating the utility of the sensor for measuring the activity of BACE1, relevant experimental parameters were optimized to enhance the performance of the sensor. To study the time-dependent cleavage of the peptide substrate by BACE1, the incubation time between the peptide modified electrode and 2.5 U/mL BACE1 was varied from 0 to 90 min. With increased incubation time, more peptides will be cleaved and less HAP-ALP probes captured by the sensor, resulting in decreased electrochemical current. However, with incubation times over 60 min the sensitivity increase levels off (Fig 5A). Thus, considering the time and sensitivity of the assay, 60 min of incubation time was chosen for the following experiment. It is also essential to be optimize the mass ratio of ALP to Aβ antibody on the HAP surface. During the HAP nanoprobe preparation process, the mass ratio of ALP to Aβ 13


0.5


antibody was varied from 1:1 to 10:1, and the sensitivity of the sensor to 2.5 U/mL of BACE1 was increased sequentially. Further increase of the ratio would lead to decreased sensitivity. This is due to the lowered binding affinity of the nanoprobe to the electrode surface due to decreased loading of Aβ antibody on HAP (Fig 5B). As a result the mass ratio of 10:1 was selected for further experiments.

A

3.0

B

3.0 2.5

2.5

Current change, µ A


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2.0 1.5

2.0 1.5 1.0 0.5

1.0 0

20

40 60 Time, min

80

0.0 100 0

5

10

15

Mass ratio of ALP to Aβ antibody

Fig. 5. Effect of BACE1 and peptide incubation time (A) and the mass ratio of ALP to Aβ antibody (B) on the sensitivity of the sensor.

In order to quantitatively test the response of the sensor to BACE1, a series of sensors were prepared for the detection of different concentrations of BACE1. As shown in Fig 6, the SWV peak current at around 0.22V was decreased with the increase of BACE1 concentrations. For the HAP-ALP sensor a good linear relationship between the current change and the logarithm of BACE1 concentrations in the range from 0.25 to 100 U/mL was attained (inset (a) of Fig. 6) while for the HAP signal (inset (b)) the linear range of the signal was around 2.5 to 25 U/mL. This compares to 0.25 to 100 U/mL of 14


HAP-ALP,

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suggesting an ALP amplification of 10X using dual signal amplification. Based on a signal-to noise of 3, the detection limit of the HAP-ALP sensor was calculated as 0.1 U/mL.

0

A

-2

h

B Current change, µA

Current, µ A

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0 1

a -10 0.1

a

6

10

100

Concentration, U/mL

0.2

0.3

0.4

0.5

E vs Ag/AgCl (V)

Fig. 6. SWV responses of the sensor to different concentrations of BACE1. Voltammagrams in A are labeled from a to h as 0. 0.25, 2.5, 5, 10, 25, 50, 100 U/mL, respectively. The inset B is the calibration curve for BACE1 based on (a) HAP-ALP as nanoprobe and (b) HAP as nanoprobe.

Sensor specificity, reproducibility and stability. To study the specificity of the sensor, the effect of several enzymes on the sensor response have been explored, including protein kinase A (PKA), alkaline phosphatase (ALP), glucose oxidase (GOx) and alcohol dehydrogenase (ADH). The experimental results indicate that at the same concentration of these enzymes, their response is low compared to that of BACE1, indicating high selectivity of the sensor (Fig 7). For sensor reproducibility, six sensors were prepared independently for the detection of 2.5 and 25 U/mL of BACE1. The relative standard deviation of the assay results were 3.4% and 3.6%, indicating good reproducibility of the 15



sensor. To test the sensor stability, the sensor was stored at 4 oC when not in use. After one month, the sensor can still retain around 85% of its original signal, indicating good stability of the sensor.

4


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3

2

1

0 BACE1

PKA

ALP

GOx

ADH

Samples

Fig. 7 Response of the sensor to different enzymes.

Detection of BACE1 Inhibition. After analyzing the analytical performance of the sensor, the sensor was evaluated using BACE1 inhibitors. Screening BACE1 inhibitors is of important because inhibition of BACE1 represents an attractive therapeutic target with the potential to slow or prevent AD. The inhibitor OM99-2, originally designed by Ghosh and Tang, was selected as a model in order to study its effect on BACE1.1 25 U/mL samples of BACE1 were treated with different concentrations of OM99-2 for 1 h and the activity of BACE1 was then measured. BACE1 activity decreased with the increase of OM99-2 concentration resulted in current increase (Fig. 8). The relationship between current intensity and concentration of OM99-2 resulted in a sigmoidal profile (inset of Fig. 8). These results clearly demonstrate that the sensor is capable of screening BACE1 inhibitors. 16


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a Current, µ A

-2 Current, µ A

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-4 -6 -8

i

-4 -5 -6 -7 -8 -9 -10

0 40 80 120 160 OM99-2 concnetration,nM

-10 0.1

0.2

0.3

0.4

0.5

E vs Ag/AgCl (V)

Fig. 8 The inhibition effect of different concentrations of OM99-2 on the response of the sensor to 25 U/mL of BACE1, curves a to i are 5, 10, 20, 40, 60, 80, 100, 120, 150 nM, respectively. The inset is the dose response curve of inhibition of BACE1 activity by OM99-2.

Detection of BACE1 in serum samples. In studying macrocyclic phosphino dipeptide isostere inhibitors of beta-secretase (BACE1), it is important to assay the stability of the peptide in human serum.

It is well known that the use of peptides as effective drugs is

often limited due to factors such as poor cellular activity, limited penetration of the blood-brain barrier, or serum instability.45

For these reasons we have assayed BACE1

activity in serum samples. A recovery test was performed by adding different concentrations of BACE1 to human serum samples. As shown in Table 1, the detected concentration of BACE1 was well matched with the amount of BACE1 added. The recovery results obtained were between 94.3 and 98.1 %, indicating that the serum matrix has a negligible effect on the detection of BACE1 by the sensor. 17



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Table 1 Recovery test of BACE1 in human serum samples

Sample No.

Amount of BACE1 detected (U/mL)

Recovery (%)

1

Amount of BACE1 added (U/mL) 2.50

2.45

98.1

2

25.0

23.6

94.3

3

50.0

48.6

97.2

4

75.0

71.0

94.6

CONCLUSION Our data suggest that ALP and Aβ antibody co-immobilized onto a HAP surface form a signal probe through reaction of phosphate groups with molybdate to form redox molybdophosphate precipitates on the electrode surface. HAP nanoparticles contained significant amount of phosphate groups, while ALP can hydrolyze pyrophosphate to produce phosphate ions. In the absence of BACE1, Aβ antibody modified HAP probe can be captured on the electrode surface due to specific binding between Aβ antibody and the peptide substrate, thereby generating a electrochemical signal. However, the cleavage of the peptide by BACE1 resulted in the detachment from electrode surface of the peptide fragment that binds to Aβ antibody, reducing the electrochemical current intensity which was demonstrated to be inversely proportional to BACE1 activity. For the first time, the reaction of HAP nanoparticles with molybdate was demonstrated to generate an electrochemical current. With the dual signal amplification strategy, the sensor displays high sensitivity and a wide linear concentration range in the BACE1 assay. Using the sensor, the effect of inhibition of OM99-2 on BACE1 activity was evaluated,

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supporting the use of the sensor for screening BACE1 inhibitors for drug development. The sensor was also used successfully for the detection of BACE1 activity in human serum samples. The sensor is simple, fast, sensitive, and can be extended to the detection of other peptidases. This dual signal enhancement method is also easily adaptable to different bioassays.

ACKNOWLEDGMENTS This work was supported by a grant from the National Natural Science Foundation of China (No. 21575165, 21375076) and the Natural Science Foundation of Hunan province (No. 2015JJ1019).

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for TOC only

Schematic illustration of dual signal amplification electrochemical sensing. The sensor is based on hydroxyapatite (HAP) nanoparticle (blue) coated with polyethylenimine (PEI) to introduce amino groups (orange) used for generating electrochemical current by the reaction of molybdate with HAP phosphate groups. The nanoparticle combines alkaline phosphatase (ALP) (purple) to generate dual signal amplification and Aβ antibody on the surface (green) to provide specificity binding of the nanoparticle to the sensor target.

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Dual Signal Amplification Electrochemical Biosensor for Monitoring

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