Bright and Stable Near-IR dyes for Bioimaging

描述(申请人提供)：光学成像是一种先进的技术，用于成像凝胶、传感器和微阵列等生物技术产品。它也越来越多地被用于监测动物的特定分子途径。后一种技术使用的荧光染料可以吸收和发射近红外(NIR)辐射(700-900 nm)，这是一个吸收血红蛋白和水最小的光谱窗口，因此允许光子穿透组织几厘米。花菁基近红外染料(Cy7)是极少数具有近红外光物理特性的商用荧光染料中最受欢迎的一种。不幸的是，它们在生物条件下非常容易降解，而且众所周知，它们会发生光化学异构化，产生非荧光产物。这一提议建立在我们最近的发现之上，即方酸轮烷(SRS)是世界上最明亮和最稳定的荧光染料之一。当前一代的SR染料显示出与Cy5染料相近的吸收和发射曲线。在这里，我们建议合成新一代近红外SR染料，它可以作为Cy7染料的优良替代品。我们的计划是开发吸收近红外的明亮和稳定的方酸轮烷染料，它可以很容易地与各种生物分子发生偶联。它们很可能成为流行的花菁(Cy7)染料的优秀替代品，并成为体外和体内成像应用的非常有用的探针。第一阶段的总体工作计划是制备新的明亮和稳定的近红外-SR染料，并比较已标记近红外-SR或Cy7的各种生物偶联物的性质。本建议的两个具体目标是：1)在MTTI上进行SA1-合成新的明亮和稳定的近红外SR NHS酯，并与3种不同类型的生物分子、锌配位的二匹卡胺配体、抗体和蛋白质进行连接，以便与Cy7类似物进行比较。第一个目标是生产明亮稳定的近红外SR染料，并根据化学稳定性和亮度确定最佳的两种。这之后将方酸轮烷探针以及作为对照探针的Cy7与锌-DPA、免疫球蛋白G蛋白和链霉亲和素偶联。2)将在UND进行SA2-生物结合物的体外评价。MTTI开发的NIR-SR和Cy7荧光免疫球蛋白和链霉亲和素生物偶联物的体外分子识别性能将使用标准凝胶电泳法和链霉亲和素/生物素方案进行表征。用荧光显微镜研究了NIR-SR-和Cy7-Zn-DPA探针与S金黄色葡萄球菌和大肠埃希氏菌的相互作用及其光稳定性。我们的目标是展示在3个不同的体系中使用标记了近红外-SR染料的分子探针与Cy7染料相比的优势。公共卫生相关性：这项研究开发的荧光探针将用于生物技术作为体外诊断设备的着色剂，并将用于医学作为活体动物疾病成像的体内探针。这种光学成像技术将帮助研究人员发现新的治疗方法，最终它将帮助医生为特定患者选择最合适的治疗方法。后一种应用是光学成像将如何促进个性化医学概念演变的一个例子。

DESCRIPTION (provided by applicant): Optical imaging is an advanced technique to image biotechnology products like gels, sensors, and micro arrays. It is also used increasingly to monitor specific molecular pathways in animals. This latter technique uses fluorescent dyes that absorb and emit near-infrared (NIR) radiation (700-900 nm) a spectral window where hemoglobin and water absorb minimally and therefore allow photons to penetrate several centimeters through tissue. Cyanine based NIR dyes (Cy7) are the popular ones among the very few commercially available fluorescent dyes showing NIR photophysical features. Unfortunately they are highly susceptible to degradation under biological conditions, and also known to undergo photochemical isomerization leading to nonfluorescent products. This proposal builds on our recent discovery that squaraine rotaxanes (SRs) are among the world's brightest and most stable fluorescent dyes. Current generation of SR dyes show absorption and emission profiles close to the Cy5 dyes. Herein we are proposing the synthesis of new generation NIR SR dyes which can be a superior substitute for Cy7 dyes. Our plan is to develop NIR absorbing bright and stable squaraine rotaxane dyes which can readily undergo conjugation with various biomolecules. They are likely to become superior replacements for the popular cyanine (Cy7) dyes and become extremely useful probes for both in vitro and in vivo imaging applications. The overall workplan for Phase I is to prepare novel bright and stable NIR-SR dyes and compare the properties of various bioconjugates that have been labeled with NIR-SR or Cy7. The two Specific Aims of the present proposal are: 1) SA1 to be performed at MTTI - Synthesize novel bright and stable NIR SR NHS esters and conjugate to 3 different types of biomolecules, a zinc coordinated dipicolylamine ligand (Zn-DPA), an antibody and a protein for comparison with Cy7 analogues. The first goal is to produce bright and stable NIR SR dyes and identifying the best two based on the chemical stability and brightness. This will be followed by conjugation of the squaraine rotaxane probes as well as Cy7 as a control probe to Zn-DPA, IgG protein and streptavidin . 2) SA2 to be perfomed at UND - In vitro evaluation of bioconjugates. In vitro molecular recognition performance of the NIR-SR and Cy7 fluorescent IgG and streptavidin bioconjugates developed at MTTI will be characterized using standard gel electrophoresis and streptavidin/biotin protocols. NIR-SR- and Cy7-Zn-DPA probes will be investigated for their interaction with S aureus and E coli bacteria membranes and their photostability using fluorescence microscopy. The goal is to demonstrate the advantage of using molecular probes labeled with an NIR-SR dye in 3 different systems compared with Cy7 dye. PUBLIC HEALTH RELEVANCE: The fluorescent probe developed by this research will be used in biotechnology as a stain for in vitro diagnostic devices, and in medical science as an in vivo probe to image disease in living animals. This optical imaging technology will help researchers discover new therapies and eventually it will help physicians choose the most appropriate therapy for a specific patient. This latter application is an example of how optical imaging will contribute to the evolving concept of personalized medicine.