Label-free electrochemical detection of enzymatic activity using peptide microarrays

人类基因组的测序提供了关于疾病遗传基础的大量有价值的信息。然而，仅仅存在3万个编码相同数量蛋白质的人类基因，并不能解释人类生命和疾病的复杂性。蛋白质的功能受到大量修饰和与其他分子的相互作用的严格调节。全面了解健康和病变细胞的细胞功能，需要了解蛋白质的功能状态。由于酶是在许多生理过程和疾病中起关键作用的蛋白质，因此测量生物样品中多种酶的活性及其与细胞功能的相关性可以提高对疾病的认识。因此，对酶活性谱的直接测量方法的需求很大。在这个项目中，我们将开发一种使用微阵列同时测量多种酶活性的新方法。这项新技术将依赖于一种简单而廉价的检测方法，即无标签电化学光谱，这将使其能够应用于即时诊断的开发。在这个项目中，我们将专注于蛋白酶，这种酶可以切割蛋白质（和较短的肽）的酰胺键。这个过程经常导致底物蛋白的激活或失活。蛋白酶活性的失调与许多疾病有关，如癌症和心血管疾病。此外，艾滋病毒和丙型肝炎等病毒编码蛋白酶，它们利用蛋白酶成功感染宿主。因此，测量生物样品（如血液）中特定蛋白酶活性谱可能有助于此类疾病的早期诊断，并有助于监测治疗过程中的进展。该项目将涉及蛋白酶底物的合成及其在金电极上的固定。我们将用电化学光谱法测量固定化底物的裂解。一旦建立了蛋白酶活性的电化学检测条件，我们将在微阵列上固定许多蛋白酶底物并同时测量多个蛋白酶活性。在未来的项目中，这种方法将被用作基于微阵列的诊断的基础，以分析生物样品（例如血液或细胞裂解物）中的蛋白酶活性。

The sequencing of the human genome has provided a wealth of valuable information regarding the genetic basis of disease. However, the existence of a mere 30.000 human genes that encode an equal number of proteins cannot explain the complexity of human life and disease. Protein function is tightly regulated by a vast number of modifications and by interactions with other molecules. A comprehensive understanding of cellular function, in both healthy and diseased cells, requires knowledge of the functional state of proteins. Because enzymes are proteins with a crucial role in many physiological processes and diseases, the measurement of multiple enzymatic activities in biological samples and their correlation with cellular function can improve the understanding disease. Therefore, methods for the straightforward measurement of enzyme activity profiles are in high demand.In this project we will develop a new method for the measurement of multiple enzymatic activities simultaneously using microarrays. The new technology will rely on a simple and cheap detection method, label-free electrochemical spectroscopy, which will enable its application in the development of point-of-care diagnostics.In this project we will focus on proteases, enzymes that cleave the amide bonds of proteins (and shorter peptides). This process often leads to the activation or inactivation of the substrate proteins. Misregulation of protease activity is linked to many diseases such as cancer and cardiovascular disease. Furthermore, viruses such as HIV and Hepatitis C encode proteases that they use to successfully infect their hosts. Therefore, the measurement of specific protease activity profiles in biological samples such as blood may assist the early diagnosis of such diseases, and help monitor progress during treatment.The project will involve the synthesis of protease substrates and their immobilisation on gold electrodes. We will measure the cleavage of the immobilised substrates by electrochemical spectroscopy. Once the conditions for the electrochemical detection of protease activity have been established, we will immobilise a number of protease substrates on a microarray and measure multiple protease activities simultaneously. In future projects, this methodology will be used as the basis for microarray-based diagnostics that profile protease activities in biological samples (e.g., blood or cell lysates).