基于磁分离及离位化学发光的核酸信息增敏自动化技术研究

Magnetic beads (MBs) can be easily used in magnetic separation and surface functionalization, so MBs are suitable choice for capturing and separating nucleic acid informations. At present the research focal spot is that the specific nucleic acid sequcence labeled with catalytic group should be captured by MBs (catalytic group-MBs, CMBs) previously modified with probes and then the chemiluminescence substrate buffer is mixed with the CMBs to produce light, the information can be read finally. In the whole procedure, there are four big problems (1) probes modified MBs would precipitate quickly because of agglomeration; (2)Steric hindrance will reduce the binding efficiency between probes on the MBs and target sequences; (3)The big chemiluminescence signal attenuation would be happened because of the shadowing effect of CMBs when CMBs will be suspended in the substeate buffer to detect the chemiluminescence information, and the quantitative examination can not be realized for the same reason; (4)Most researches, using MB as carrier, did not realize the information of auto caputuing, the roboticized feasibility of MBs could not be reflected. In this research arm molecules, linking MBs and probes, would be screened. DNA fragments obtained by multiplex polymerase chain reaction (PCR) will be captured by the probes. After the catalytic center, durable chemiluminescence substrates would be added one by one, ultrasonic will be used to break arm molecules. Consequently the centre of luminescence will be released from MBs and off site detection of chemiluminesent signal will be achieved. Finally this method will be integrated in the workstation for the automation of sample processing and information acquisition. It is our aim to develop a roboticized, high throughput, multisite, quantifiable technology for nucleic acid information enhancing sensitivity.

磁珠易于实现磁分离和功能化，适于核酸信息的捕获与分离。目前的研究热点是在磁珠表面修饰上核酸探针，捕获催化基团标记的特异核酸序列，加入化学发光底物混匀成悬浊液后读取化学发光信息，但(1)结合核酸探针的磁珠易团聚而快速沉淀；(2)修饰在磁珠表面上的核酸探针与目标序列结合时具有空间位阻；(3)直接结合了催化基团发光中心的磁珠在悬浮后检测化学发光，光信号会由于磁珠的遮蔽作用而极大衰减，因此亦不易实现定量检测；(4)目前大多数研究都没能实现自动化信息获取，未能体现磁珠的自动化可操作性。本研究拟筛选出连接磁珠与核酸探针的手臂分子，捕获特异多重PCR扩增产物，结合催化中心，加入长效化学发光底物，通过超声方式断裂手臂分子，游离出结合在磁珠上的发光中心，实现化学发光信号的离位获取，并在工作站上实现样本处理及信息获取自动化，发展出一种自动化、高通量、多位点、可定量的核酸信息增敏检测技术。

磁珠易于实现磁分离和功能化，适于核酸信息的捕获与分离。目前的研究热点是在磁珠表面修饰上核酸探针，捕获催化基团标记的特异核酸序列，加入化学发光底物混匀成悬浊液后读取化学发光信息，但(1)结合核酸探针的磁珠易团聚而快速沉淀；(2)修饰在磁珠表面上的核酸探针与目标序列结合时具有空间位阻；(3)直接结合了催化基团发光中心的磁珠在悬浮后检测化学发光，光信号会由于磁珠的遮蔽作用而极大衰减，因此亦不易实现定量检测；(4)目前大多数研究都没能实现自动化信息获取，未能体现磁珠的自动化可操作性。本研究筛选出了连接磁珠与核酸探针的手臂分子，捕获特异多重PCR扩增产物，结合催化中心，加入长效化学发光底物，通过核酸酶降解带有发光中心的核酸，游离出结合在磁珠上的碱性磷酸酶，实现了化学发光信号的离位获取，检测灵敏度达到50 fM，提高了2个数量级。课题组还尝试在微流控芯片上实现样本处理及信息获取自动化，发展出了一种适于自动化的、高通量、多位点核酸信息增敏检测技术。