Photonic Crystal Fiber Probe Fluorescence Biosensing

描述(申请人提供)：使用荧光探针来识别癌症特征和癌细胞的生物活性的方法前景广阔。基于荧光共振能量转移(FRET)和荧光相关光谱(FCS)等技术的探针提供了识别特定RNA或蛋白质分子的能力，这些分子可以识别癌症并提供肿瘤细胞使用的致癌途径的信息。其他探针可以通过测量化疗和放射诱导的细胞凋亡来洞察药物反应。然而，荧光分析有几个局限性。大多数体外分析使用流式细胞仪或复杂的共聚焦显微镜进行分析，这需要将组织从体内取出，通常分解成细胞，然后以静态方式固定和分析。体内荧光分析的问题更大，因为背景荧光和组织散射，即使在近红外范围内，也限制了对皮肤的信号采集。双光子激发是光学领域的一个重要进展，它促进了FRET、FCS和CARS技术的发展。然而，这些应用受到使用这些技术所必需的复杂技术(共聚焦显微镜)的限制。我们已经演示了通过光纤的双光子荧光分析用于分析SCID小鼠体内的癌细胞和体内的人类肿瘤。这项先前的工作构成了R21提案的等价物，因为我们实现了我们的主要目标：开发传感系统的光学和电子学，并记录该系统在体外和体内获取和分析荧光信号的能力。这项R33应用的主要目标是开发一种基于新型双包层光子晶体光纤(DCPCF)的更灵敏的原型设备，我们假设这种光纤将提供使用几种荧光技术进行体内荧光信号临床评估所需的灵敏度和冗余。我们计划以三个具体目标进行研究： 1.研制用于双光子光纤荧光探头(D-TPOFF)的DCPCF 2：利用D-TPOFF在体外对癌症信号进行量化，并使用体内和体外的靶向纳米颗粒监测肿瘤细胞中的药物效应。 3：利用D-TPOFF改用其他荧光技术来检测体内肿瘤事件。 在这些研究结束时，这项技术将达到可以商业化的地步。

DESCRIPTION (provided by applicant): Methods using fluorescent probes to identify cancer signatures and biological activities of cancer cells hold great promise. Probes based on fluorescence resonance energy transfer (FRET) and techniques such as fluorescence correlation spectroscopy (FCS) and other similar technologies offer the ability to identify specific RNA or protein molecules that can identify a cancer and provide information on oncogenic pathways used by the tumor cells. Other probes can give insight into drug response by measuring apoptosis induction by chemotherapies and radiation. However, fluorescent analysis has several limitations. Most ex vivo analyses use a flow cytometer or complex, confocal microscope to perform analyses, and this requires that tissue be removed from the body and often disrupted into cells, then fixed and analyzed in a static manner. The problems with in vivo fluorescent analysis are even greater since background fluorescence and tissue scattering, even in the near-infrared range, limit signal acquisition to the skin. Two-photon excitation has been a critical advance in optics, facilitating FRET, FCS and CARS techniques in vitro. However, these applications are limited by the complex technology (confocal microscopy) necessary to employ these techniques. We have demonstrated the use of two-photon fluorescence analysis through optical fibers for analysis of cancer cells in vitro and human tumors in vivo in SCID mice. This prior work constitutes the equivalent of an R21 proposal, as we achieved our major objectives: to develop sensing system optics and electronics and to document the ability of this system to obtain and analyze fluorescence signals in vitro and in vivo. The primary goal of this R33 application is to develop a more sensitive prototype device based on a novel dual-clad photonic crystal fiber (DCPCF) that we hypothesize will provide the sensitivity and redundancy necessary for the clinical evaluation of fluorescence signals in vivo using several fluorescence techniques. We plan to carry out our studies in three Specific Aims: 1: Develop DCPCF for use in a two-photon optical fiber fluorescence probe (D-TPOFF). 2: Utilize the D-TPOFF to quantify cancer signatures in vitro and monitor drug effects in tumor cells using targeted nanoparticles ex vivo and in vivo. 3: Utilize D-TPOFF to adapt other fluorescent techniques to examine events in tumors in vivo. At the end of these studies, this technology will be at a point where it is ready for commercialization.