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

描述（由申请人提供）：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是重要的，因此可以促进癌症的早期检测。今天，大多数用于检测生物分子的方法都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记检测器，提供可扩展性（尺寸减小，传感器数量增加），用于检测多种分析物。然而，大多数微/纳米传感器尚未达到基于标记的检测方案的灵敏度水平。该提案探索了一套新的方法，以提高荧光/无放射性标记平台的灵敏度，从而能够在临床显著条件和浓度下检测癌症标志物。该提案的具体目标集中在将变构核酸催化剂（适体酶）适应于1）纳米机械悬臂梁生物传感器，这是一种模型无标记平台，近年来引起了学术界的广泛关注，但由于灵敏度不足，尚未获得太多临床认可，2）基于珠子的衍射生物传感器，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上的固定化底物（在被靶蛋白激活后）的适体酶将基于其在表面上的功能性（Aim 1）开发，并适于与悬臂和基于珠的衍射生物传感器（Aim 2）一起操作。对于悬臂梁传感器，由靶分子激活的大适体酶（通过引入额外的核苷酸或通过将适体酶缀合到微米尺寸的颗粒来构建）将连接到功能化的悬臂梁表面并产生放大的信号。对于衍射生物传感器，用适体酶功能化的磁珠将用于从复杂混合物中选择性地捕获目标分子。捕获将激活适体酶并允许它们连接（并因此连接珠）到以交替模式功能化的固体表面。珠子的结合将导致产生非常敏感的信号的固体衍射光栅。两个平台中的进一步信号增强将通过进行滚环扩增来实现。预期通过适体酶检测蛋白质将产生比通过直接检测非共价蛋白质-受体结合产生的信号大得多的信号。两种不同的癌症相关蛋白质将被用作模型靶点，针对其开发适体酶：碱性成纤维细胞生长因子（bFGF）和血管内皮生长因子（VEGF）。预计将这些新的适体酶适应纳米机械悬臂梁传感器和新的基于珠的衍射生物传感器将使bFGF和VEGF的检测限达到50 fM和500 fM。将在具有生物学意义的培养基（如血清和细胞裂解物）中检测所开发的策略（目标3）。适体酶的开发将在德克萨斯大学奥斯汀分校进行，而所有实验工作将由普渡大学承担。许多癌症如果早期发现是可以治疗的。检测体液中的癌症标志物是实现早期检测的一种有前途的方法。该项目将成为开发灵敏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.