利用离子敏感场效应晶体管检测从干血斑提取的样品中的α-半乳糖苷酶 A 反应

Alexander Kuznetsov, Andrey Sheshil, Eugene Smolin, V. Grudtsov, D. Ryazantsev, Mark Shustinskiy, Tatiana Tikhonova, Irakli Kitiashvili, Valerii Vechorko, Natalia Komarova
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摘要

法布里病是一种溶酶体贮积症,由α-半乳糖苷酶A的活性显著降低或缺失引起。由于酶替代疗法的出现,新生儿筛查中对法布里病的诊断是合理的。本文介绍了一种使用互补金属氧化物半导体(CMOS)兼容离子敏感场效应晶体管(ISFET)和氧化铪敏感表面检测干血斑提取物中α-半乳糖苷酶 A 活性的电化学方法。结果表明,ISFET 能够检测由 α-半乳糖苷酶 A 催化的反应。对缓冲液成分进行了优化,以便为酶和 ISFET 的性能提供合适的条件。使用 ISFET 结构作为传感器元件,可以无标记地检测α-半乳糖苷酶 A 的天然底物三聚木糖的酶促反应,而不是合成的含氟底物。ISFET 芯片与印刷电路板和微流体反应室封装在一起,可使用定制装置进行长期信号测量。实验证明,封装后的传感器可以区分干血斑提取物中正常和受抑制的 GLA 活性。所述方法为扩大法布里病新生儿筛查的分布范围提供了一种前景广阔的解决方案。
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Detection of α-Galactosidase A Reaction in Samples Extracted from Dried Blood Spots Using Ion-Sensitive Field Effect Transistors
Fabry disease is a lysosomal storage disorder caused by a significant decrease in the activity or absence of the enzyme α-galactosidase A. The diagnostics of Fabry disease during newborn screening are reasonable, due to the availability of enzyme replacement therapy. This paper presents an electrochemical method using complementary metal-oxide semiconductor (CMOS)-compatible ion-sensitive field effect transistors (ISFETs) with hafnium oxide-sensitive surfaces for the detection of α-galactosidase A activity in dried blood spot extracts. The capability of ISFETs to detect the reaction catalyzed by α-galactosidase A was demonstrated. The buffer composition was optimized to provide suitable conditions for both enzyme and ISFET performance. The use of ISFET structures as sensor elements allowed for the label-free detection of enzymatic reactions with melibiose, a natural substrate of α-galactosidase A, instead of a synthetic fluorogenic one. ISFET chips were packaged with printed circuit boards and microfluidic reaction chambers to enable long-term signal measurement using a custom device. The packaged sensors were demonstrated to discriminate between normal and inhibited GLA activity in dried blood spots extracts. The described method offers a promising solution for increasing the widespread distribution of newborn screening of Fabry disease.
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