Quantitative molecular profiling of tumor biomarkers with multi-color monovalent

描述（由申请人提供）：随着癌症靶向治疗的发展，在肿瘤穿刺活检中建立准确定量分析多种肿瘤生物标志物的方法势在必行。准确有效地捕获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，并且正在挑战目前基于过氧化物酶的免疫组织化学方法，该方法不是化学计量学，只能在一个切片上进行一次染色。纳米技术正在成为癌症研究进步的基本驱动力。在这方面，生物共轭量子点（QDs）由于其高复用和量化能力，作为一种新兴的肿瘤生物标志物分析探针特别有吸引力。与有机染料和荧光蛋白相比，量子点具有较高的亮度和光稳定性，可以在单一激发波长下同时激发多种荧光颜色。虽然量子点已经成为一种新的生物标记实体，但精确控制每纳米颗粒中生物分子的数量和生物分子的取向的“完美”量子点生物偶联物仍然缺乏，迫切需要准确定量地检测生物标志物的表达。缺乏定向控制会导致qd配体活性的丧失，而缺乏化学计量控制会导致信号量化的变化。提出的研究目标是利用混合凝胶电泳技术开发一组多色取向控制的单价量子点探针。由于量子点不能进入或被聚丙烯酰胺凝胶分离，该凝胶体系具有很强的分离量子点的能力。虽然琼脂糖凝胶的孔径比杂化凝胶大，可以使量子点进入，但琼脂糖凝胶中量子点的分离无法达到杂化凝胶的分离分辨率。一种含有两个抗原结合位点的抗体，含有一个抗原结合位点的单价抗体是利用还原剂对整个抗体进行部分还原而产生的。偶联取向控制是通过位点特异性生物素化抗体铰链区域的巯基来实现的，并使抗原结合位点保持完整。每个量子点与一个链霉亲和素拷贝的偶联物和单价量子点将通过混合凝胶分离，并使用实验室制造的洗脱装置回收。使用福尔马林固定和石蜡包埋（FFPE）前列腺癌细胞系遵循标准免疫组织化学程序。采用高光谱成像系统进行数据采集和定量分析。新型单价量子点探针将为确定个体癌症的分子指纹图谱提供前所未有的精密工具，从而做出准确的诊断和治疗决策。我们的结果将与NIH的使命相关，并对研究各种疾病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.