双信号放大电化学生物传感器，用于监测阿尔茨海默氏症相关蛋白酶β-秘密酶的活性和抑制作用,Analytical Chemistry

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双信号放大电化学生物传感器，用于监测阿尔茨海默氏症相关蛋白酶β-秘密酶的活性和抑制作用
Analytical Chemistry ( IF 6.7 ) Pub Date : 2016-10-17 00:00:00 , DOI: 10.1021/acs.analchem.6b02659
Fengli Qu _{1,

2} , Minghui Yang ₁ , Avraham Rasooly ₃

Affiliation

蛋白酶BACE1（β位淀粉样蛋白前体蛋白裂解酶1）催化合成β-淀粉样蛋白（Aβ）的第一步，β-淀粉样蛋白是在阿尔茨海默氏病（AD）中在大脑中蓄积的肽。BACE1活性的测量对于开发BACE1抑制剂来减缓或终止AD至关重要。为了测量电极固定的底物肽的BACE1裂解，我们开发了一种生成氧化还原的羟基磷灰石（HAP）探针，该探针通过纳米粒子与钼酸盐（MoO ₄^2–）。该探针将用于双重信号放大的碱性磷酸酶（ALP）与Aβ抗体相结合，将探针与电极表面上固定的肽底物结合。我们在0.25至100 U / mL的浓度范围内测量了BACE1的活性。与仅使用HAP的探头相比，使用双信号HAP-ALP探头将信号增加了一个数量级，从而使检测限低至0.1 U / mL。为了测量对BACE1活性的抑制，将BACE1抑制剂OM99-2添加到25 U / mL的BACE1中，浓度范围为5至150 nM。观察到的抑制检测极限为OM99-2为10 nM。这些结果证明了这种新型生物传感器测量BACE1活性和BACE1活性抑制剂的能力。据我们所知，这是第一个关于HAP纳米粒子与钼酸盐反应可产生电化学电流的报道。这种双重信号放大策略可以扩展到其他电化学分析中，并适用于广泛的应用。

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Dual Signal Amplification Electrochemical Biosensor for Monitoring the Activity and Inhibition of the Alzheimer’s Related Protease β-Secretase

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 (MoO₄^2–). 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 to 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 wide applications.

更新日期：2016-10-17

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