Label-free detection of cancer markers using aptazyme-based amplification

描述(申请人提供)：以快速和灵敏的方式检测癌症标志物对于确定疾病的类型和阶段非常重要，因此可以促进癌症的早期发现。今天，大多数用于检测生物分子的方法使用荧光和放射性标记，这增加了分析所需的时间、成本和专业知识。微米和纳米技术使研究人员能够开发无标签检测器，为检测多种分析物提供可扩展性(减小尺寸和增加传感器数量)。然而，大多数微/纳米传感器还没有达到基于标记的检测方案的灵敏度水平。这项建议探索了一套新的方法来提高荧光/无放射性标签平台的灵敏度，以便能够在临床上有意义的条件和浓度下检测癌症标记物。该提案的具体目标集中在将变构核酸催化剂(Aptazyme)应用于1)纳米机械悬臂式生物传感器，这是一种近年来引起极大关注但由于灵敏度不足而没有得到临床接受的模型无标记平台，以及2)基于微珠的衍射生物传感器，这是一种更新的检测无荧光平台。可以共价结合到固体表面的固定化底物上的Aptazyme(在被目标蛋白激活时)将基于它们在表面上的功能而被开发(Aim 1)，并被改造成与悬臂和基于微珠的衍射生物传感器一起操作(Aim 2)。对于悬臂传感器，被目标分子激活的大的aptazyme(通过引入额外的核苷酸或通过将aptazyme连接到微米大小的颗粒上而构建)将连接到功能化的悬臂表面并产生放大的信号。对于衍射式生物传感器，将使用带有aptazyme功能化的磁珠来选择性地从复杂混合物中捕获目标分子。捕获将激活端粒酶，并允许它们连接(从而连接珠子)到以交替模式功能化的固体表面。珠子的结合将产生产生非常敏感信号的固体衍射光栅。两个平台的信号将通过滚圈放大实现进一步的增强。与通过直接检测非共价蛋白质-受体结合产生的信号相比，通过aptazyme检测蛋白质预计将产生更大的信号。两种不同的癌症相关蛋白将被用作开发aptazyme的模型靶点：碱性成纤维细胞生长因子(BFGF)和血管内皮生长因子(VEGF)。预计将这些新的aptazyme应用于纳米机械悬臂梁传感器和基于微珠的新型衍射生物传感器将使bFGF和VEGF的检测下限分别达到50fM和500fM。开发的策略将在具有生物学意义的介质中进行测试，如血清和细胞裂解物(目标3)。端粒酶的开发将在奥斯汀的德克萨斯大学进行，而所有的实验工作将由普渡大学进行。如果及早发现，许多癌症是可以治疗的。检测体液中的癌症标志物是实现早期检测的一种很有前途的方法。该项目将成为开发敏感但相对简单的癌症标志物检测平台的里程碑。

DESCRIPTION (provided by applicant): Detection of cancer markers in a fast and sensitive fashion is important in determining the type and stage of the disease and hence can facilitate early detection of cancer. Today, most methods used to detect biomolecules utilize fluorescent and radiolabels, which increases the time, cost and expertise required for assays. Micro- and nanotechnology has enabled researchers to develop label-free detectors that offer scalability (down in size and up in number of sensors) for detecting multiple analytes. However, the majority of micro/nanosensors have not yet achieved the sensitivity levels of label-based detection schemes. This proposal explores a novel set of methods to enhance the sensitivity of fluorescent/radio-label-free platforms to enable detection of cancer markers in clinically significant conditions and concentrations. The specific aims of the proposal focus on adapting allosteric nucleic acid catalysts (aptazymes) to 1) a nanomechanical cantilever biosensor, which is a model label-free platform which has attracted an immense amount of academic attention in recent years but has not received much clinical acceptance due to insufficient sensitivity, and 2) a bead-based diffraction biosensor, which is a much newer detection fluorescence-free platform. Aptazymes that can covalently bind to immobilized substrates on a solid surface (upon activation by target proteins) will be developed based on their functionality on a surface (Aim 1) and adapted for operation with the cantilever and the bead-based diffraction biosensor (Aim 2). For the cantilever sensor, large aptazymes (constructed either by introducing additional nucleotides or by conjugating aptazymes to micron-sized particles) activated by target molecules will ligate to the functionalized cantilever surface and yield an amplified signal. For the diffraction biosensor, magnetic beads functionalized with aptazymes will be used to selectively capture target molecules from complex mixtures. The capture will activate the aptazymes and allow their ligation (and hence the ligation of the beads) to a solid surface that is functionalized in an alternating pattern. The binding of the beads will result in a solid diffraction grating that generates very sensitive signals. Further signal enhancement in both platforms will be achieved by performing rolling circle amplification. It is expected that detection of proteins via aptazymes will generate far greater signals in comparison with those generated by direct detection of non-covalent protein-receptor binding. Two different cancer-relevant proteins will be used as model targets against which aptazymes will be developed: basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). It is expected that adapting these new aptazymes to the nanomechanical cantilever sensor and the new bead-based diffraction biosensor will enable 50 fM and 500 fM detection limits for bFGF and VEGF. The developed strategy will be tested in biologically significant media such as serum and cell lysate (Aim 3). The aptazyme development will be performed at The University of Texas at Austin while all experimental work will be undertaken by Purdue University. Many cancers can be treated if detected early. Detecting cancer markers in body fluids is a promising way to achieve early detection. This project will constitute a milestone in development of sensitive but relatively simple platforms for detection of cancer markers.