Holt Brandon Alexander, Mac Quoc D, Kwong Gabriel A
Wallace H. Coulter Department of Biomedical Engineering, Georgia Tech College of Engineering and Emory School of Medicine.
Wallace H. Coulter Department of Biomedical Engineering, Georgia Tech College of Engineering and Emory School of Medicine; Parker H. Petit Institute of Bioengineering and Bioscience; Institute for Electronics and Nanotechnology, Georgia Tech; Integrated Cancer Research Center, Georgia Tech; The Georgia Immunoengineering Consortium, Emory University and Georgia Tech;
J Vis Exp. 2018 Jul 16(137):57937. doi: 10.3791/57937.
Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.
蛋白酶是多功能酶，专门负责肽键的水解，并控制包括体内平衡和应激稳态在内的广泛生物过程。此外，蛋白酶活性失调会引发疾病，并且是癌症等疾病的功能性生物标志物；因此，在体内检测蛋白酶活性的能力可为生物医学诊断提供临床相关信息。本方案的目标是创建纳米传感器，通过在尿液中产生可量化信号来探测体内的蛋白酶活性。这些蛋白酶纳米传感器由两部分组成：纳米颗粒和底物。纳米颗粒的作用是延长循环半衰期，并将底物递送至目标疾病部位。底物是一个短肽序列（6 - 8个氨基酸），设计成对特定的目标蛋白酶或一组蛋白酶具有特异性。底物与纳米颗粒表面结合，并由一个报告基团（如荧光标记）终止，用于检测。当失调的蛋白酶切割肽底物时，报告基团被过滤到尿液中进行定量，作为蛋白酶活性的生物标志物。在此，我们描述了一种用于基质金属蛋白酶9（MMP9）的纳米传感器的构建，MMP9与肿瘤进展和转移相关，用于在小鼠模型中检测结直肠癌。
