Field effect sensing for protein microarrays

蛋白质微阵列的场效应传感

• 批准号: BB/D523094/1
• 负责人: Jason Davis
• 金额: $ 123.11万
• 依托单位: University of Oxford
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD523094%2F1
• 关键词: Field; effect; sensing; protein; microarrays

We are made up of trillions of cells, and each cell is made of billions of proteins. To understand how our bodies work, we need to understand how the proteins in a cell interact. The first step in this understanding was the sequencing of the human genome, the set of genes whose DNA encodes the proteins we wish to understand. This has allowed us to design probes- in some cases using human DNA itself- that can be printed in very high densities on microscope slides. We can print a probe that is specific for every gene onto a single microscope slide, and so ask which genes are being turned on, or off to make RNA or not, at every stage in the life of a cell. This ability has already created a new understanding of biology, and led to a new way of thinking about how cells work. Though impressive, this still paints but a very incomplete picture of cellular machineries at work, because although they tell us a lot about the expression of normal genes as RNA, they do not tell us anything about whether or not protein is made from that RNA (it isn¿t always), nor do they tell us about the expression of proteins whose genes carry mutations that will lead to disease. To rectify this, we would need to print probes that recognise all of the different forms of expressed proteins. This has been attempted, using either antibodies or human proteins themselves as the probes. While this makes a lot of sense, because both antibodies and proteins have evolved to recognise specific isoforms of each protein, it has proved difficult, for two reasons. First, because antibodies and proteins are very fragile, printing them onto glass slides causes them to lose their ability to recognise their target proteins in cell extracts. Second, proteins are minute, and detecting the interaction between two is correspondingly hard. So far, the best way of doing this is to attach a dye to the proteins in cell extracts, so that the printed protein lights up if it recognises its target protein in the extract. This is problematic: attaching the dye can change the protein, and we run the risk that it may no longer be recognised by our probes or may even be recognised by the wrong ones. We plan to address each of these difficulties. First, we have access to libraries of artificial antibodies that have the ability to recognise cellular proteins, but are very stable and can be printed onto a slide without losing this ability. We know that this is true for the first ones we have tried, and part of this proposal will allow us to ask why this works, and whether we can learn from the behaviour of these proteins ideas that will help us with other, less stable proteins. Second, we plan to expand upon our finding that when proteins are printed on the surface of an electrode, they change the electrical properties of the electrode in a measurable way. Then, if a second protein (from a cell extract) binds to the first protein, we get another change- so if we have many electrodes, each carrying one receptor protein, we will be able to measure the binding of each cell protein in turn, and thus the behaviour of and interactions between, every protein in a cell at any given time. This will allow us to gain intimate insights into the molecular basis of life in a cell.

我们由数万亿个细胞组成，每个细胞由数十亿个蛋白质组成。要了解我们的身体是如何工作的，我们需要了解细胞中的蛋白质是如何相互作用的。这一理解的第一步是人类基因组的测序，这组基因的DNA编码我们希望了解的蛋白质。这使我们能够设计探针-在某些情况下使用人类DNA本身-可以在显微镜载玻片上以非常高的密度打印。我们可以在一张显微镜载玻片上打印一个对每个基因都有特异性的探针，这样就可以知道在细胞生命的每个阶段，哪些基因被打开或关闭来制造RNA。这种能力已经创造了对生物学的新理解，并导致了对细胞如何工作的新思考方式。虽然令人印象深刻，但这仍然描绘了一幅非常不完整的细胞机制工作的画面，因为尽管它们告诉我们很多关于正常基因作为RNA表达的信息，但它们并没有告诉我们蛋白质是否由RNA组成（它并不总是），也没有告诉我们基因携带突变的蛋白质的表达，这些突变会导致疾病。为了纠正这一点，我们需要打印识别所有不同形式表达蛋白质的探针。这已经尝试过了，使用抗体或人类蛋白质本身作为探针。虽然这很有意义，因为抗体和蛋白质都已经进化到可以识别每种蛋白质的特定亚型，但事实证明这很困难，原因有两个。首先，由于抗体和蛋白质非常脆弱，将它们打印到载玻片上会导致它们失去识别细胞提取物中目标蛋白质的能力。其次，蛋白质是微小的，检测两者之间的相互作用也相应地很难。到目前为止，最好的方法是将染料附着在细胞提取物中的蛋白质上，这样打印的蛋白质就会在识别提取物中的目标蛋白质时发光。这是有问题的：附着染料可以改变蛋白质，我们冒着这样的风险，它可能不再被我们的探针识别，甚至可能被错误的探针识别。我们计划解决这些困难中的每一个。首先，我们可以获得人工抗体库，这些抗体具有识别细胞蛋白质的能力，但非常稳定，可以打印到载玻片上而不会失去这种能力。我们知道，对于我们尝试的第一种蛋白质来说，这是正确的，这个提议的一部分将使我们能够问为什么它会起作用，以及我们是否可以从这些蛋白质的行为中学习到有助于我们研究其他不太稳定的蛋白质的想法。其次，我们计划扩展我们的发现，即当蛋白质被打印在电极表面时，它们会以可测量的方式改变电极的电特性。然后，如果第二种蛋白质（来自细胞提取物）与第一种蛋白质结合，我们就会得到另一种变化-所以如果我们有许多电极，每个电极携带一种受体蛋白质，我们将能够依次测量每种细胞蛋白质的结合，从而测量细胞中每种蛋白质在任何给定时间的行为和相互作用。这将使我们能够深入了解细胞中生命的分子基础。