Luminometric Nanoparticle-Based Assay for High Sensitivity Detection of β-Amyloid Aggregation.
Luminometric Nanoparticle-Based Assay for High Sensitivity Detection of β-Amyloid Aggregation.
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DOI：
10.1021/acs.analchem.6b04266
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发表时间：
2017-02
影响因子：
7.4
通讯作者：
S. Pihlasalo;T. Deguchi;M. Virtamo;Jenna Jacobino;K. Chary;F. López-Picón;Gerda Brunhofer-Bolzer;Roope J Huttunen;A. Fallarero;P. Vuorela;H. Härmä
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文献类型：
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作者：
S. Pihlasalo;T. Deguchi;M. Virtamo;Jenna Jacobino;K. Chary;F. López-Picón;Gerda Brunhofer-Bolzer;Roope J Huttunen;A. Fallarero;P. Vuorela;H. Härmä
开发了一种利用时间分辨发光共振能量转移（TR-LRET）的基于纳米颗粒的测定方法，用于检测β-淀粉样蛋白聚集。该测定是基于竞争吸附的样品和受体标记的蛋白质供体铕（III）聚苯乙烯纳米粒子。通过跟踪β-淀粉样肽1-42（Aβ42）随时间变化的纤维化，并与参考方法原子力显微镜（AFM）和硫磺素T（ThT）测定进行比较，证明了该测定的性能。纤维化导致Aβ42对纳米颗粒的吸附减少，增加了TR-LRET信号。所研究的方法检测原纤维形成具有相同的灵敏度。对文献中报道的8种潜在的浮选抑制剂化合物进行了测试，并对每种方法获得的结果进行了比较。AFM成像显示，任何化合物对原纤维形成的抑制都不完全。所开发的TR-LRET纳米颗粒测定给出了与AFM成像相对应的结果。然而，ThT测定导致矛盾的结果，因为在所有测试化合物的存在下测量到低荧光信号，表明对荧光化的抑制。我们的研究结果表明，开发的TR-LRET纳米颗粒测定可用于筛选潜在的β-淀粉样蛋白聚集抑制剂，而一些测试的化合物可能被测量为假阳性抑制剂与经常使用的ThT测定。
A nanoparticle-based assay utilizing time-resolved luminescence resonance energy transfer (TR-LRET) was developed for the detection of β-amyloid aggregation. The assay is based on the competitive adsorption of the sample and the acceptor-labeled protein to donor europium(III) polystyrene nanoparticles. The performance of the assay was demonstrated by following the fibrillization of β-amyloid peptide 1-42 (Aβ42) as a function of time and by comparing to the reference methods atomic force microscopy (AFM) and thioflavin T (ThT) assay. The fibrillization leads to reduced adsorption of Aβ42 to the nanoparticles increasing the TR-LRET signal. The investigated methods detected fibril formation with equal sensitivities. Eight potential fibrillization inhibitor compounds reported in the literature were tested and the results obtained with each method were compared. It was shown with AFM imaging that the inhibition of fibril formation was not complete with any of the compounds. The developed TR-LRET nanoparticle assay gave corresponding results with the AFM imaging. However, the ThT assay led to contradictory results, as low fluorescence signal was measured in the presence of all tested compounds suggesting inhibition of fibrillization. Our results suggest that the developed TR-LRET nanoparticle assay can be exploited for screening of potential β-amyloid aggregation inhibitors, whereas some of the tested compounds may be measured as false positive inhibitors with the much-utilized ThT assay.