Development of a monitoring system of biochemical tumor markers during surgery operation

在这项研究中，我们开发了一种基于表面等离子体共振(SPR)的探针式生物传感器用于生化肿瘤标志物的检测系统。在本研究期间，我们开发了探针式生物传感器，并尝试以C反应蛋白(CRP)为例进行检测，作为肿瘤标记物的例子。结果表明，该生物传感器可以检测5-g/m1的C反应蛋白。利用这一探针，我们还开发了一种葡萄糖传感探针，因为葡萄糖在肿瘤周围存在高浓度。为此，采用酶显色法对葡萄糖敏感反应的中间产物过氧化氢进行检测。实验结果表明，我们能够成功地研制出检测过氧化氢的酶显色SPR生物传感器探针。体外筛选出与肿瘤周围氧化应激标志物8-羟基-2‘-脱氧鸟苷(8-OHdG)结合的DNA适配子。对测序的24个DNA适配子的序列分析表明，所有克隆都含有一个长度为26个核苷酸的保守序列。具有保守序列的DNA探针可以与SPR传感器探针结合应用于肿瘤周围组织的氧化应激状态的评估。最后，我们开发了与MRI图像匹配的新标记，并尝试了传感器探针的位置。这种标记物被称为混合标记物，具有两种功能：一是用于磁共振成像的造影剂，二是用于位置测量的反射球。体模测试表明，混合标记应放置在距离接收线圈50 mm的区域内。按常规标记物对既往临床病例的分析表明，平面角度40°~69°占80%。

In this research we have developed a detection system for biochemical tumor markers with a probe type biosensor based on surface plasmon resonance(SPR). We expect that the biosensor can be applied to a rapid detection of tumors during the surgery by inserting the probe around the affected area.In this research period, we have developed a probe type biosensor and attempted to measure C-reactive protein(CRP), as an example of the tumor markers. It was found that the biosensor can detect 5 □g/m1 of CRP. Using this probe, we have also developed a glucose sensing probe because glucose exists at high concentration around tumor. For this, an enzyme-chromogenic method was applied to detect hydrogen peroxide, which is intermediate of glucose sensing reaction. As the results, we could successfully develop the enzyme-chromogenic SPR biosensor probe for hydrogen peroxide detection.In vitro selection of DNA aptamers which bind to8-Hydroxy-2'-deoxyguanosine(8-OHdG), one of oxidative stress markers around tumor, was demonstrated. Sequence analysis of sequenced 24 clones of the DNA aptamers revealed that all clones contained a conserved sequence with 26 nucleotides length. A DNA probe with the conserved sequence could be applied to the evaluation of oxidative stress of tissues around the tumor in combination with SPR sensor probes.At last, we developed new markers to match the MRI image and the position of the sensor probe was attempted. The markers, named Hybrid marker , had two functions ; one was a contrast agent for MRI, the other was a reflective ball for position measurement. A phantom test showed that the hybrid markers should place inside the area of 50mm far from the receiver coil. An analysis for the past clinical cases by conventional marker exhibited that an angle of the plane contained 80% cases from 40 to 69 degrees.