Digital Microfluidic Platform for Multiplexing Enzyme Assays: Implications for Lysosomal Storage Disease Screening in Newborns
Digital Microfluidic Platform for Multiplexing Enzyme Assays: Implications for Lysosomal Storage Disease Screening in Newborns
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DOI：
10.1373/clinchem.2011.163139
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发表时间：
2011-10-01
影响因子：
9.3
通讯作者：
Pamula, Vamsee K.
中科院分区：
文献类型：
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作者：
Sista, Ramakrishna S.;Eckhardt, Allen E.;Pamula, Vamsee K.
背景：由于酶替代疗法的可用性，新生儿溶酶体贮积病（lsd）筛查已获得相当大的兴趣。我们提出了一个数字微流控平台来执行快速，多重酶分析酸性-葡萄糖苷酶（GAA）和酸性-半乳糖苷酶筛选Pompe和Fabry疾病。结果与标准荧光法所得结果进行了比较。方法:我们在杜克生物化学遗传学实验室对60个已鉴定的新生儿干血斑（DBSs）进行了基于实验室的荧光酶分析，外加10个受pompee影响的样本和11个受fabry影响的样本，每次检测使用3毫米穿孔，培养时间为20小时。我们在Advanced Liquid Logic Inc.使用数字微流控平台自动化荧光酶分析，使用单个穿孔的提取物进行两种检测。孵育时间为6小时。孵育时间为1小时，也进行了检测。结果：检测结果大致相当，尽管使用微流体的GAA平均酶活性比使用基于实验室的方法高约3倍，这可能归因于更高的底物浓度。使用数字微流体在6和1小时的孵育时间内观察到正常样品和受影响样品之间明显的分离。结论：与临床参考实验室相比，数字微流控平台更有利于对Pompe和Fabry疾病的DBSs进行酶分析。该平台为新生儿筛查实验室提供了一种新技术，通过在廉价的系统中完全自动化所有液体处理操作来筛选lsd，并提供快速结果。(C) 2011美国临床化学学会
BACKGROUND: Newborn screening for lysosomal storage diseases (LSDs) has been gaining considerable interest owing to the availability of enzyme replacement therapies. Wepresent a digital microfluidic platform to perform rapid, multiplexed enzymatic analysis of acid alpha-glucosidase (GAA) and acid alpha-galactosidase to screen for Pompe and Fabry disorders. The results were compared with those obtained using standard fluorometric methods.METHODS: We performed bench-based, fluorometric enzymatic analysis on 60 deidentified newborn dried blood spots (DBSs), plus 10 Pompe-affected and 11 Fabry-affected samples, at Duke Biochemical Genetics Laboratory using a 3-mm punch for each assay and an incubation time of 20 h. We used a digital microfluidic platform to automate fluorometric enzymatic assays at Advanced Liquid Logic Inc. using extract from a single punch for both assays, with an incubation time of 6 h. Assays were also performed with an incubation time of 1 h.RESULTS: Assay results were generally comparable, although mean enzymatic activity for GAA using microfluidics was approximately 3 times higher than that obtained using bench-based methods, which could be attributed to higher substrate concentration. Clear separation was observed between the normal and affected samples at both 6- and 1-h incubation times using digital microfluidics.CONCLUSIONS: A digital microfluidic platform compared favorably with a clinical reference laboratory to perform enzymatic analysis in DBSs for Pompe and Fabry disorders. This platform presents a new technology for a newborn screening laboratory to screen LSDs by fully automating all the liquid-handling operations in an inexpensive system, providing rapid results. (C) 2011 American Association for Clinical Chemistry