Integrated Fiber Optic Sensor for DNA Hydridization

描述(由申请人提供)：本提案的目标是开发一种多路复用、便携式实时消逝光纤传感器仪器，用于定量样品中特定DNA的数量。在该项目的第一阶段，IA演示了使用带有荧光检测的消逝光纤DNA生物传感器实时监测DNA杂交的可行性。DNA生物传感器安装在温度控制的反应室中。利用聚合酶链式反应(PCR)扩增DNA序列，并与固定在DNA生物传感器表面的特定探针杂交。在第二阶段，IA将开发一种多路DNA生物传感器荧光仪，能够同时监测八个或更多个DNA生物传感器上发生的杂交。与消逝传感相关的灵敏度的提高将允许在25-30个周期后检测到一个DNA拷贝。仪器的小尺寸和相关的样品准备盒将使其适用于用于病原体鉴定的护理点测试。 具体目的1规定在仪器方法中加入探针，使用内部聚合酶链式反应标准的探针，以使每个单独反应的效率标准化。特定目标2提供了一个多路复用传感器盒，使得每个盒可以同时运行八个样品。具体目的3提供了原型仪器的性能与已建立的使用含有HPV16或沙眼衣原体特异序列的质粒的方法的比较。 将在特定目标4下开发一种用于样品制备的专用试剂盒。这将使具有临床意义的生物样品无需离心机即可制备用于聚合酶链式反应。样本将被涂在试剂盒的顶部，试剂将用注射器压出。DNA将被洗脱，准备在易于携带的传感器仪器中进行放大。当仪器记录八种溶液和标准的杂交率时，将在传感器盒中原位进行聚合酶链式反应。这将提供预期七对数范围内的对照样本和标准曲线。 该系统可以适应多种扩增形式，并有可能用于遗传学研究实验室和医院实验室的临床检测。它将提供一种独特的仪器和样品制备方法，适用于在短时间内对病原体进行护理点检测。

DESCRIPTION (provided by applicant): The goal of this proposal is development of a multiplexed, portable real time evanescent fiber optic sensor instrument for quantifying the amount of specific DNA in a sample. During Phase I of this project, IA demonstrated the feasibility of monitoring DNA hybridization in real time using an evanescent fiber-optic DNA biosensor with fluorescence detection. The DNA biosensor was mounted within a temperature controlled reaction chamber. DNA sequences were amplified using the polymerase chain reaction (PCR) and hybridized to specific probes immobilized to the DNA biosensor surface. During Phase II, IA will develop a multiplexed DNA Biosensor Fluorometer capable of simultaneously monitoring the hybridization occurring on eight or more DNA biosensors at a time. The increased sensitivity associated with evanescent sensing will allow detection of one DNA copy after 25-30 cycles. The small size of the instrument and associated sample prep cartridge will adapt it to point of care testing for pathogen identification. Specific Aim 1 provides for incorporation into the instrumental methods, use of a probe for an internal PCR standard for normalization of the efficiency of each individual reaction. Specific Aim 2 provides a multiplexed sensor cartridge so that each cartridge can run eight samples simultaneously. Specific Aim 3 provides comparison of performance of the prototype instrument with established methods using plasmids containing sequences which are specific for HPV 16 or for Chiamydia trachomatis. A specialized cartridge for sample preparation will be developed under Specific Aim 4. This will allow biological samples of clinical interest to be prepared for PCR without the need of a centrifuge. Sample will be applied to the top of the cartridge and reagents will be pressed through with a syringe. DNA will be eluted ready for amplification in the easily portable sensor instrument. PCR will be performed in situ in the sensor cartridge while the instrument records the rate of hybridization for eight solutions and standards. This will provide for a control sample and a standard curve over an anticipated seven log range. The system can be adapted to a variety of amplification formats, and has the potential for use in genetics research laboratories and for clinical determinations in hospital labs. It will provide an instrument and sample preparation method uniquely adapted to point of care testing for pathogens in a short period of time.