利用苯硼酸衍生物作為晶片表面修飾並應用於人體血清中登革熱病毒檢測

Abstract

登革熱為一個特定區域的疾病。登革熱病毒被發現在埃及伊蚊和白紋伊蚊傳染給哺乳動物並有機會造成出血性登革熱導致死亡率增加的情況。如何避免登革熱傳播與疾病感染，早期階段之檢測為主要研究主題之一。我們的研究將重點放在感染早期的檢測上。石英晶體微量天平（QCM）是一個較佳的偵測平台，以檢測生物分子之間的相互作用，例如抗原與抗體或者適配體與蛋白質...等。我們選擇這個平台，主要利用壓電效應來檢測分析物的質量，因為QCM的靈敏度高並提供微流體系統。QCM晶片使用標準化清洗方式來清潔金表面的氧化物殘留。首先我們先合成一個含有硫醇基之苯硼酸化合物，簡稱APBA-MUA。該連接子可控制抗體修飾於QCM晶片表面之位向，其反應原理是藉由APBA-MUA可以直接與抗體之醣基化位點結合，而達成抗體定向固定化的目標。 研究中，我們選擇登革病毒1型（DV1）作為偵測目標。可以檢測到100〜200 CFU / ml的DV1中的病毒液。現在，我們試圖使在人血清的檢測包含病毒粒子和減少非特異性結合的QCM系統。並且為了減少QCM晶片的使用量，我們合成了具有特定官能團的PBA連接子，它可以高度與硫醇基團反應。在金表面形成二硫化物與硼酸連接子作用連接，它可以藉由三(2-羰基乙基)磷鹽酸鹽(TCEP)把表面清除乾淨。最後，我們成功地展示了這種在QCM系統平台，適用於登革熱病毒檢測。

The Quartz crystal microbalance (QCM) is a useful platform for detecting the interactions between bio-molecules such as antigen/antibody, aptamer/protein…etc. For promoting the detection sensitivity of QCM and reduce the cost from chip comsumption, we synthesized the interface linker by coupling the aminophenylboronate with the carboxylic residue of the 2-thiolpyridine-capped 11-mercaptoundecanoic acid. The phenylboronic acid residue was used for controlling the orientation of antibody on chip through the complex formation of boronic acid with sugar moiety on antibody. In order to establish the chip regeneration process, ethylene dithiol was coated on gold chip surface through the self-assembly manner. The thiol on chip surface was then employed to replace the thiolpyridine moiety of the linker to form a disulfide bond, which can be broken down by treating with tris(2-carboxyethyl)phosphine. The experimental results revealed that the sensor chip can be reused, with minor loss of sensitivity, for at least 4 times. Quantitative assessment on Dengue virus 1 (DV1) detection was performed using this new strategy. 1% N-laurylsarcosine (LSS) was further added in a serum sample (with 100-fold dilution) to eliminate non-specific interaction of chip and molecules in serum. Results showed that the detection exhibited a good linear co-relation in the range of 1000 to 1,000,000 cfu/ml of DV1.