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的便携式真实的时间瞬逝光纤传感器仪器，用于定量样品中特定DNA的量。在该项目的第一阶段，IA证明了使用具有荧光检测的倏逝光纤DNA生物传感器在真实的时间内监测DNA杂交的可行性。将DNA生物传感器安装在温度受控的反应室内。使用聚合酶链反应（PCR）扩增DNA序列，并与固定在DNA生物传感器表面的特异性探针杂交。在第二阶段，IA将开发一种多重DNA生物传感器荧光计，能够同时监测8个或更多个DNA生物传感器上发生的杂交。与瞬逝感测相关的增加的灵敏度将允许在25-30个循环后检测一个DNA拷贝。仪器和相关样本制备盒的小尺寸将使其适合用于病原体识别的护理点测试。 具体目标1规定将探针用于内部PCR标准品，以标准化每个单独反应的效率，并将其纳入仪器方法。Specific Aim 2提供了一个多路传感器盒，每个盒可以同时运行8个样本。具体目标3提供了原型仪器与使用含有HPV 16或沙眼衣原体特异性序列的质粒的已建立方法的性能比较。 将在具体目标4下开发用于样品制备的专用检测盒。这将允许临床感兴趣的生物样品在不需要离心机的情况下制备用于PCR。将样品加到检测盒顶部，用注射器压入试剂。DNA将被洗脱，准备在易于携带的传感器仪器中进行扩增。将在传感器盒中原位进行PCR，同时仪器记录8种溶液和标准品的杂交速率。这将提供对照样品和预期7个对数范围内的标准曲线。 该系统可以适应各种扩增形式，并有可能用于遗传学研究实验室和医院实验室的临床测定。它将提供一种仪器和样品制备方法，特别适用于在短时间内对病原体进行即时检测。

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.