Point of Care CARD Device for Rapid Molecular Diagnostics

描述（由申请人提供）：该项目将推进床旁（POC）分子诊断设备的开发，用于在非传统卫生保健环境中快速、廉价地检测引起传染病的病原体。现有的完全集成的微流体化学和试剂装置（CARD（tm））将被优化用于检测性传播感染（STI）和尿路感染（UTIs）中的病原体。CARD（tm）能够自动分离和裂解感染因子，然后提取和纯化DNA和/或RNA，并PCR扩增和检测靶序列（迄今为止，全血、血浆、唾液和阴道拭子已被成功处理）。为了提高现有CARD（tm）的易用性，该项目将进一步整合使用稳定的脂质体（已证明至少三年的真实的时间稳定性），以增加信号强度，同时将生物信号转换为电子信号。这将通过将与扩增的靶序列的部分互补的寡聚核苷酸探针缀合至脂质体的外表面来实现，脂质体的外表面进而将包封电活性化合物。随后，通过使用与靶序列的另一区域互补的其它寡核苷酸探针缀合的磁珠，将所得脂质体-靶复合物磁性固定在CARD（tm）的微流体通道内。在固定的脂质体裂解后，释放的电活性化合物将被允许流到叉指型超微电极阵列的表面上，在那里连续且可逆的氧化还原反应将产生与分析物的浓度成正比的电流。因此，该项目将允许不同程度的培训（包括不熟练）的个人进行复杂的分子诊断，因为一旦样品被引入，所有的制备，分析和读出功能都将自动执行。

DESCRIPTION (provided by applicant): This project will advance the development of a point-of-care (POC) molecular diagnostic device for the rapid and inexpensive detection of infectious disease-causing pathogens in nontraditional heath care settings. An existing, fully integrated microfluidic Chemistry and Reagent Device (CARD(tm)) will be optimized for detection of pathogens in sexually transmitted infections (STIs) and urinary tract infections (UTIs). The CARD(tm) is capable of automatically isolating and lysing infectious agents, then extracting and purifying DNA and/or RNA and PCR amplifying and detecting target sequences (to date, whole blood, plasma, saliva and vaginal swabs have been successfully processed). To improve the ease-of-use of the existing CARD(tm), this project will further integrate the use of stable liposomes (at least three year real time stability has been demonstrated) to increase the signal intensity and simultaneously transduce the biological signal into an electronic one. This will be accomplished by conjugating oliogonucleotide probes that are complementary to portions of the amplified target sequences, to the exterior surface of liposomes, which, in turn, will encapsulate electroactive compounds. The resulting liposome-target complexes will be subsequently magnetically immobilized within the microfluidic channels of the CARD(tm) by use of magnetic beads conjugated to other oligonucleotide probes that are complementary to another region of the target sequences. Upon lysis of the immobilized liposomes, the released electroactive compounds will be allowed to flow onto the surface of an interdigitated ultramicroelectrode array where a continuous and reversible redox reaction will generate electric current that will be directly proportional to the concentration of the analyte. This project will therefore allow sophisticated molecular diagnostics to be performed by individuals of varying degrees of training (including unskilled), since once the sample is introduced all preparative, analytical and readout functions are automatically performed.