使用附自動採樣匣之高分子鍍覆石英晶體微天平構築免疫反應型腫瘤胚胎抗原生物感測器

目的：惡性腫瘤居103年度國人十大死因之首位，其中，結腸直腸癌、胃癌、與食道癌等消化道癌症，分佔第三位、第七位與第九位。腫瘤胚胎抗原（CEA）是人類消化道癌症的腫瘤標記，被廣泛應用於消化道癌症之篩檢，目前多採用免疫分析法進行檢測，但是檢測過程需耗時2-3小時。因此，本研究擬開發可即時檢測腫瘤胚胎抗原之免疫反應型生物感測器。材料與方法：生物感測分析技術被譽為二十一世紀分析及篩檢科技之新寵，係由抗體、酵素等生物識別性材料，配合微質量天平、電化學電極等各種訊號轉換器組合而成。本研究利用腫瘤胚胎抗原單株抗體對腫瘤胚胎抗原之高度專一性與親和性，配合具備高安定性、高敏感度、即時輸出、重複使用等多項優點之石英晶體微質量天平（quartz crystal microbalance, QCM）元件作為訊號轉換器，同時配合自行開發之自動採樣裝置以及以聚對二甲苯（poly-parylene）對QCM元件進行表面鍍覆製成pQCM之技術，共同組合構成能夠即時檢測腫瘤胚胎抗原濃度以便即時篩檢消化道癌症之免疫反應型腫瘤胚胎抗原生物感測器。結果：本研究將腫瘤胚胎抗原單株抗體固定在pQCM元件表面構成的腫瘤胚胎抗原感測器，對溶解在PBS緩衝液分別配製成0.5、2.5、25以及200 ng/ml濃度之腫瘤胚胎抗原標準樣品溶液進行檢測，對於低濃度（0.5 ng/ml）及高濃度（200 ng/ml）樣品，僅分別需時5分鐘及少於10分鐘即可完成檢測操作；在8天內重複進行16次檢測操作，相對振頻移差都能夠維持在95% 以上，顯示其具備操作安定性。相對於腫瘤胚胎抗原樣品，對濃度同樣為25 ng/ml之含鐵蛋白（ferritin）以及甲型胎兒蛋白（α-fetoprotein），僅分別有3.9% 及4.6%之應答強度（responsiveness）；對同時包含濃度分別為25 ng/ml之含鐵蛋白與甲型胎兒蛋白之25 ng/ml腫瘤胚胎抗原樣品，檢測數據僅比單獨對25 ng/ml腫瘤胚胎抗原樣品之應答（responsiveness）增加6.5%，證實其具備優異專一性。結論與討論：本研究除可完成本論文之腫瘤胚胎抗原感測器之開發外，所建立之系統技術亦可應用於其他腫瘤標記生物感測器之開發，並可更進一步發展可同時檢測多項腫瘤標記之集積式多功能癌症篩檢系統，對於癌症之早期發現提供重要貢獻。

關鍵字

磁振導航聚焦超音波熱治療；日常品質確保；位置穩定性

並列摘要

Purpose : This study aims at developing an immune-biosensor that can detect carcinoembryonic antigens (CEAs) in real time. Malignant tumors were the top cause of death in Taiwan in 2014. Among the GI tract cancer deaths, colorectal cancer, gastric cancer, and esophageal cancer ranked third, seventh, and ninth. CEAs are the tumor biomarkers used when screening for human GI tract cancers. At present, immunoassays are the most widely used screening method. However, they are extremely time-consuming and require approximately 2-3 h per test. Thus, the present study is aimed at developing an immune-biosensor that can perform real-time CEA detection. Materials and Methods : Biosensor analysis technologies are fast becoming the preferred technology for analysis and screening in the 21st century. Such technologies operate by combining biometric materials, such as antibodies and enzymes, and various signal converters, such as microbalances or electrochemical electrodes. In the present study, a quartz crystal microbalance (QCM), with advantages of high stability, high sensitivity, real-time output, and reusability, was coated with polypropylene and equipped with a self-developed auto-sampling device to create a novel polypropylene-coated quartz crystal microbalance with auto-sampling (pQCM). The pQCM was then combined with CEA-related antibodies (high specificity and affinity to CEAs) to develop an immuno-biosensor that can instantaneously measure CEA concentrations, thereby creating an effective means to instantly screen for gastrointestinal cancers. Result : CEA-related monoclonal antibodies were adhered to the surface of the pQCM to create a CEA sensor. Four standard sample solutions were prepared by dissolving 0.2, 2.5, 25, and 200 ng/ml of CEA into phosphate buffered saline solution. The CEA sensor was then used to test the standard sample solutions. The samples with the lowest (0.5 ng/ml) and highest (200 ng/ml) CEA concentrations required only 5 and 10 min to complete testing, respectively. Tests were repeated 16 times over 8 days. Results indicated that the relative vibration frequency shift was maintained at 95% or higher, suggesting that the results achieved excellent operational stability. For comparison, samples prepared with 25 ng/ml of ferritin and α-fetoprotein only achieved a responsiveness of 3.9% and 4.6%, respectively. Subsequently, the test results of the 25 ng/ml CEA samples separately prepared with the 25 ng/ml of ferritin and α-fetoprotein showed an increase in responsiveness of only 6.5% compared to those of the pure 25 ng/ml CEA sample, confirming the excellent specificity of the proposed biosensor. Discussion : The proposed CEA sensor satisfies the requirements of the present study; in addition, the systems technology established in the present study can be applied to developing biosensors for multiple cancer biomarkers or even a cumulative and multifunctional cancer screening system, greatly benefiting the early identification of cancer.

並列關鍵字

Biosensor ； Quartz crystal microbalance (QCM) ； Poly-parylene quartz crystal microbalance (pQCM)

國際替代計量

使用附自動採樣匣之高分子鍍覆石英晶體微天平構築免疫反應型腫瘤胚胎抗原生物感測器

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