Development of rapid analysis device for periodontal disease activity

包括前胶原I型C-末端前肽（PICP）在内的几种生物标志物已被报道用于客观评价牙周病的状态。虽然这些标志物通常用常规的ELISA方法用酶标仪检测，但该方法耗时且样品量大，并且不适用于自动分析仪。因此，需要用于测量生物标志物的方便、灵敏和准确的方法。生物纳米技术具有快速、高通量、操作简便、样品和试剂用量少、微型化和自动化等特点，在分析化学领域引起了广泛的关注。为了开发口腔临床牙周病生物标志物的分析系统，我们首先在微芯片上开发了基于微通道内抗原抗体反应的定量分析系统。在环烯烃共聚物（COC）微芯片上的微通道（300 μm宽，100 μm深）表面上吸附第一抗PICP抗体后，将样品和过氧化物酶缀合的第二抗PICP抗体混合物引入微通道中30分钟，随后洗涤并测量过氧化物酶活性以测定PICP水平。双对数图分析表明，在50 ~ 600 ng/ml范围内，PICP浓度与过氧化物酶活性呈线性关系（r^2=0.9745）。用本方法测定了4例受试者体液中PICP浓度，并与常规ELISA法测定结果进行了比较。经一元线性回归分析，各方法间有良好的相关性（P<0.05）。我们证明了微芯片用于检测PICP水平的可能性，具有高灵敏度和准确性，以及牙科临床诊断的潜力。

Several biomarkers including procollagen type I C-terminal propeptide(PICP), have been reported for objective evaluation of the state of periodontal disease. Although these markers are usually examined by conventional ELISA method with a microplate reader, it is time-and sample-consuming, and it is not adaptable to automated analyzers. Convenient, sensitive, and accurate methods for the measurement of the biomarkers are thus required. Bio-nano technology has received considerable interest in analytical chemistry due to the intrinsic characteristics of high speed, high throughput, easy operation, low consumption of samples and reagents, miniaturization, and automation. To develop the analysis system of biomarkers in periodontal disease in dental clinic, we first developed the quantitative analysis system based on the antigen-antibody reaction in microchannel on microchip. After the adsorption of 1st anti-PICP antibody on the surface of microchannel (300 μm width, 100 μm depth) on cyclic olefin copolymers (COC) microchip, sample and peroxidase-conjugated 2nd anti-PICP antibody mixture was introduced into a microchannel for 30 min, followed by washing and peroxidase activity was measured for the determination of PICP level. A double-logarithm plot analysis revealed a linear relationship between PICP concentration and peroxidase activity in the range of 50 to 600 ng/ml of PICP (r^2=0.9745). PICP concentrations in humoris of four subjects determined by the present methods were compared with the results obtained by conventional ELISA method. Good correlations were observed for each method on simple linear regression analysis (P<0.05). We demonstrated the possible application of microchip for determination of PICP levels with high sensitivity and accuracy, and the potential for the dental clinical diagnosis.