Quantitative molecular profiling of tumor biomarkers with multi-color monovalent

描述(申请人提供)：随着癌症靶向治疗的发展，建立在针状肿瘤活检中准确和定量分析多个肿瘤生物标志物的方法是势在必行的。准确而有效地捕捉每个患者肿瘤的特定分子肖像在癌症的诊断和治疗中起着至关重要的作用，这对目前基于过氧化物酶的免疫组织化学方法提出了挑战，这种方法不是化学计量的，一张切片上只能进行一次染色。纳米技术正在成为癌症研究进步的根本驱动力。在这方面，生物共轭量子点(QD)作为一种新兴的肿瘤生物标记物分析探针，由于其高度的多路复用和定量能力而特别吸引人。与有机染料和荧光蛋白相比，量子点具有独特的优势，如高亮度和光稳定性，用一个激发波长同时激发多种荧光颜色。尽管量子点已经成为一种新的生物标记实体，但精确控制每纳米粒子的生物分子数量和生物分子的取向的完美的量子点生物结合物仍然是不可获得的，迫切需要准确和定量的检测生物标记物的表达。缺乏方向控制会导致QD配体活性的丧失，而缺乏化学计量控制会导致信号定量的变化。这项研究的目标是利用混合凝胶电泳法开发一种多色取向可控的单价量子点探针。这种凝胶系统能够很好地分离量子点，因为量子点不能进入，也不能被聚丙烯酰胺凝胶分离。虽然量子点可以进入琼脂糖凝胶，因为它的孔径比杂化凝胶大，但量子点在琼脂糖凝胶中的分离不能达到杂化凝胶的相同分辨率。抗体包含两个抗原结合部位，含有一个抗原结合部位的单价抗体是通过使用还原剂部分还原整个抗体而产生的。结合方向控制是通过抗体铰链区的位点特异性生物素化巯基实现的，并保持抗原结合部位不变。每一天一份链霉亲和素和一价一次的链霉亲和素的结合物将通过杂化凝胶分离，并使用实验室制造的淋洗设备回收。使用福尔马林固定和石蜡包埋(FFPE)的前列腺癌细胞株将遵循标准的免疫组织化学程序。将应用超光谱成像系统进行数据采集和定量分析。新的单价QD探针将在确定单个癌症的分子指纹方面提供前所未有的精确工具，据此可以做出准确的诊断和治疗决定。我们的结果将与NIH的使命相关，并对研究各种疾病的病理学的研究人员产生广泛的兴趣。 公共卫生相关性：本项目将利用正在进行的一组多色单价量子点探针的合成来进行肿瘤生物标记物的分析，准确定量测量肿瘤标记物的预测结果将为癌症诊断和个性化治疗提供必要的信息。我们的研究将与美国国立卫生研究院的使命相关，并对研究各种疾病的病理学的研究人员产生广泛的兴趣。

DESCRIPTION (provided by applicant): With the development of targeted therapies in cancer, it is imperative to establish methods for analyzing multiple tumor biomarkers accurately and quantitatively in needle tumor biopsies. Accurately and efficiently capturing a specific "molecular portrait'' of each patient's tumor plays a crucial role in the diagnosis and treatment cancers and are challenging the current method of peroxidase-based immunohistochemistry which is not stoichiometric and only one stain can be done on a section. Nanotechnology is becoming a fundamental driver of advances in cancer research. In this regard, bioconjugated quantum dots (QDs) are particularly attractive as an emerging new probe for tumor biomarker profiling owing to its high multiplexing and quantification capabilities. QDs possess unique advantages compared to organic dyes and fluorescent proteins, such as high brightness and photostability, simultaneous excitation of multiple fluorescence colors with a single excitation wavelength. Although QDs have become a new biological labeling entity, the "perfect '' QD bioconjugates, in which the number of biomolecules per nanoparticle and the orientation of the biomolecules are precisely controlled, are still not available and pressing in need for accurate and quantitative detection biomarker expression. The lack of orientation control can cause the loss of QD-ligand activity while the lack of stoichiometric control will bring about variation in signal quantification. The goal of the proposed research is to develop a panel of multi-color orientation controlled monovalent QD probes using hybrid gel electrophoresis. This gel system is highly capable of separating QDs because QDs cannot enter or be fractionated by poly-acrylamide gel. Although QDs can enter agarose gel due to its larger pore size than hybrid gel, QD separation in agarose gel cannot reach the same resolution as hybrid gel can. An antibody contains two antigen binding sites, monovalent antibody containing one antigen binding site is generated by partial reduction of a whole antibody using a reducing agent. Conjugation orientation control is realized through site-specific biotinylating sulfhydryls in the antibody hinge region and leave the antigen binding site intact. Conjugates with one copy of streptavidin per QD and monovalent QD will be separated through the hybrid gel and recovered using a lab-made eluter device. The standard immunohistochemistry procedures will be followed using formalin-fixed and paraffin-embedded (FFPE) prostate cancer cell lines. Hyperpectral imaging system will be applied to perform data acquisition and quantitative analysis. Novel monovalent QD probes will provide an unprecedented precision tool in determining the molecular fingerprints of individual cancers, on which accurate diagnosis and treatment decision can be made. Our results will be relevant to the mission of the NIH and be broadly interesting to researchers studying pathology of a variety of diseases. PUBLIC HEALTH RELEVANCE: This project will conduct tumor biomarker profiling by using ongoing synthesis of a panel of multi-color monovalent Quantum Dot probes, the predicting results of accurate quantitative measurement tumor markers will provide the essential information for cancer diagnosis and personalized treatment. Our research will be relevant to the mission of the NIH and be broadly interesting to researchers studying the pathology of variety diseases.