Multiplexed Protein Mapping Using Nanopores and DNA Technology for Cancer Risk Stratification

癌症仍然是最具挑战性的疾病之一，它可以遗传到肿瘤内的异质性。肿瘤内异质性可以定义为个体患者肿瘤内部的变化。了解这种异质性可以通过检测单个肿瘤细胞中的不同蛋白质来提供肿瘤蛋白质谱，这也被称为蛋白质制图。纳米孔传感是一种很有前途的方法，可以提供多种蛋白质的特异性和敏感性图谱。纳米孔传感是基于电阻式脉冲传感，其中一个纳米孔被用来连接两个有电解质溶液的腔室。当施加电荷时，电解质穿过纳米孔，产生基线电流。这种力可以用来驱动分子从一个腔室到另一个腔室，这将导致部分阻塞，从而引起电流下降。电流随时间的下降与分子的大小和电荷相对应。这可以用来绘制和量化DNA上的结构。具有蛋白质特异性结合位点的DNA分子可以用来识别某些蛋白质的存在，因为每个蛋白质-蛋白质-结合位点复合物都会导致特定DNA位置的电流下降。这些DNA结构可以被称为DNA载体。在这里，我们打算设计每个载体上至少有40个不同蛋白质结合位点的DNA载体，以改进目前可以识别多达40种蛋白质的技术；比如单细胞条形码芯片和飞行时间细胞术。为了区分每个DNA载体，每个DNA载体一端嵌入的不同DNA结构可以作为条形码来识别不同的DNA载体及其相应的蛋白质。这样做的目的是绘制癌症特异性蛋白质的图谱。这也带来了自身的挑战，特别是在纳米孔测量过程中，蛋白质结合城在高盐浓度下的结合稳定性。通过使用DNA-蛋白质交联、适体结合、DNA-抗体偶联物或DNA序列修饰，将确保稳定的蛋白质结合。为了确保数据的临床可靠性，将从潜在合作者处获得定义明确的肺癌细胞和食道细胞。已建立的单细胞分离和回收技术将与磁分离相结合，从单个细胞中回收DNA载体。通过DNA载体的蛋白质谱分析将允许细胞特异性蛋白质景观的量化。该项目的结果将有助于提供一种临床可靠的风险分层生物传感方法，该方法可以帮助优化诊断并确定针对不同癌症类型的最佳治疗或干预方案。

Cancer is still one of the most challenging diseases which can be inherited to its intra-tumour heterogeneity. Intra-tumour heterogeneity can be defined as the changes within cancer in individual patient. Understanding this heterogeneity can be tackled by detecting different proteins in individual tumour cells to provide a tumour protein profile, which is also known as protein mapping. A promising approach to provide a specific and sensitive mapping of multiple proteins is nanopore sensing. Nanopore sensing is based on resistive pulse sensing, where a nanopore is used to connect two chambers that have electrolyte solution. When a charge is applied, electrolytes move through the nanopore producing a base line current. This force can be used to drive molecules from one chamber to another, which would result in a partial blocking that induces a drop in current. The drop in current over time corresponds to the size and the charge of the molecule. This can be used to map and quantify structures on the DNA. DNA molecule with protein specific binding sites across its length can be used to identify the presence of certain proteins as each of the protein-protein-binding-site complex would result in a drop in current at a specific DNA location. These DNA constructs can be refereed as DNA carriers. Here we intend to design DNA carriers with at least 40 different protein binding sites on each carrier to improve upon current technologies that can identify up to 40 proteins; such as single-cell barcode chips and time-of-flight mass cytometry. To differentiate between each DNA carrier, different DNA structures embedded on one end of each DNA carrier can be used as barcoded to identify the different DNA carriers and their corresponding proteins.The aim of this is to enable mapping of the cancer-specific proteins. This comes with its own challenges, especially the stability of the binding in high salt concentration between the protein binding cites during the nanopore measurements. Stable protein binding will be ensured by the use of DNA-protein crosslinking, aptamer binding, DNA-antibody conjugates, or DNA sequence modification.To ensure the clinical reliability of the data, well-defined lung cancer cells and oesophagus cells from a potential collaborator will be obtained. Established single-cell isolation and recovery techniques in combination with magnetic separation are to be done to recover DNA carrier from individual cells. The analysis of the protein profiles via the DNA carrier will allow for the quantification of cell-specific proteins landscape.The outcome of this project will help in providing a clinically reliable risk stratification biosensing method that can aid in optimizing diagnostics and deciding the best course of treatment or intervention for different cancer types.