Microassays for Enzyme Activity

酶活性微量测定

• 批准号: 6643930
• 负责人: Wilson Harvey Smart
• 金额: $ 40.11万
• 依托单位: KUMETRIX, INC.
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-04-15 至 2005-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6643930
• 关键词: analytical method; bioassay; bioengineering /biomedical engineering; biomedical equipment development; cholinesterases; computer simulation; enzyme activity; hydrolase; oxidoreductase; technology /technique development

DESCRIPTION (provided by applicant): Longitudinal metabolic and physiological studies in small animal models of disease will greatly benefit from new methods of easy, rapid enzyme assay in very small sample volumes (sub-microliter). Phase 1 demonstrated the feasibility of adapting silicon microsampling and assay devices, originally developed for blood glucose monitoring, to enzyme activity measurement. A novel technique, "Diffusive Enzyme Activity Measurement" was developed. An electrochemical redox-mediated serum cholinesterase assay was the example, requiring no on-chip biologicals (providing a potentially long shelf-life). Silicon microchips with novel diffusional microfluidics were designed and fabricated. These disposable microchips have integrated silicon microneedles capable of penetrating the skin and taking a blood sample painlessly and reliably. In Phase II, this technique will be generalized to other enzymes. A computer simulation model will be used, experimental work will develop assays for the families of cholinesterases and lactate oxidizing enzymes, and a prototype readout instrument will be built. The enzymes chosen for Phase ll kinetic constants are covering many orders of magnitude, to probe the range of applicability of this new analytical method. Potential commercial applications include enzyme activity measurements on sub-microliter liquid samples, such as blood drawn from experimental animals (or humans), tissue extract specimens, purified enzymes, and drug discovery research and development.

描述（由申请人提供）：在小动物疾病模型中进行的纵向代谢和生理学研究将大大受益于在非常小的样品体积（亚微升）中进行简单、快速酶测定的新方法。第1阶段证明了将最初为血糖监测而开发的硅微取样和测定装置用于酶活性测量的可行性。提出了一种新的酶活性测定技术--扩散法。电化学氧化还原介导的血清胆碱酯酶测定是一个例子，不需要芯片上的生物制剂（提供一个潜在的长保质期）。设计并制作了具有新型扩散微流体的硅微芯片。这些一次性微芯片集成了硅微针，能够穿透皮肤，无痛可靠地采集血液样本。在第二阶段，这项技术将推广到其他酶。将使用计算机模拟模型，实验工作将开发胆碱酯酶和乳酸氧化酶家族的测定方法，并将建立一个原型读出仪器。选择用于阶段II动力学常数的酶涵盖许多数量级，以探索这种新分析方法的适用范围。潜在的商业应用包括对亚微升液体样品的酶活性测量，例如从实验动物（或人类）抽取的血液、组织提取物标本、纯化的酶以及药物发现研究和开发。