以表面電漿子共振法偵測唾液中腫瘤標記

Detection of Tumor Marker in Human Saliva by Surface Plasmon Resonance Methods

梁楊鴻(Yang-Hung Liang)

指導教授 ： 林啟萬

國立臺灣大學/工學院/醫學工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.02945

摘要

本論文主要研究目的為以兩種不同的表面電漿子共振法(surface plasmon resonance (SPR)) 來感測唾液中的腫瘤標記分子carbohydrate antigen 15-3 (CA15-3) 。血清中CA15-3的測量可以運用在許多不同類型的癌症，特別是在乳癌方面，常用來輔助診斷乳癌的判定，治療效果的評估，治療後的追蹤等等，一直是乳癌腫瘤標記中的黃金標準(golden standard) ，人體唾液中也被發現含有微量的CA15-3，和血清中濃度有正相關之關係，同時由於唾液能反應人體即時的狀態，可以推測其運用價值可能高於血清。量測唾液中的CA15-3有幾個優點:1. 取得容易.2. 可重覆測試3. 反應患者即時的狀態。但也有幾個困難度:1. 唾液取得若無嚴格限制取得條件，其生理性變異度將可能造成錯誤結果.2. 唾液中的CA15-3濃度比血清低10倍以上，偵測不易3. 市面上尚無唾液CA15-3標準品等相關商品，必需自行製造。4. 唾液的分析前處理研究尚未清楚。本研究針對上述困難點試圖提出可行之方法 ，希望對未來唾液偵測有所貢獻。研究中以透析置換方式成功製造唾液CA15-3標準品, 並使用實驗室開發的高靈敏金/氧化鋅奈米薄膜之表面電漿共振晶片系統，同時比較已商品化之Biacore公司出產之X-100型表面電漿共振系統，來評估唾液CA15-3的偵測結果。研究結果發現，以高靈敏金/氧化鋅奈米薄膜之表面電漿共振晶片系統偵測唾液CA15-3，其線性範圍在2.5–20 U/mL (乳癌病人判定值約為4 U/mL).，Biacore公司出產之X-100型表面電漿共振系統則40–300 U/mL.。依過去Streckfus實驗室研究資料顯示, 以本實驗室開發高靈敏金/氧化鋅奈米薄膜之表面電漿共振晶片系統來偵測唾液CA15-3, 其線性範圍已可涵蓋並分辨病人乳癌之分期。在動力學的資料方面，Biacore公司出產之X-100型表面電漿共振系統KD值為381 U/mL而金/氧化鋅奈米薄膜之表面電漿共振晶片系統則為17 U/mL; 這明顯差異可能來自於Biacore公司所使用的晶片含dextran膠所造成的立體屏障和阻力所造成。本研究發現以高靈敏金/氧化鋅奈米薄膜之表面電漿共振晶片系統可以成功地偵測低濃度之唾液CA15-3，不需進行額外濃縮程序。期盼未來這套系統能再精研, 可在居家或臨床快速獲取測試結果。同時本研究也提供唾液偵測的基礎資訊，希望對有興趣唾液偵測的研究人員有所裨益和啟發。

關鍵字

表面電漿共振法 ； 唾液偵測 ； 腫瘤標記分子CA15-3 ； 高靈敏金/氧化鋅奈米薄膜表面電漿共振系統 ； Biacore X-100型表面電漿共振系統

並列摘要

The main purpose in this dissertation is to measure the presence of the tumor marker carbohydrate antigen 15-3 (CA15-3) in human saliva using two different surface plasmon resonance (SPR) systems. To compare the sensitivity of an SPR biosensor based on thin-film Au/ZnO and the Biacore SPR system. CA15-3 is a tumor marker in human cancers, especially for the diagnosis of breast cancer recurrence and monitoring. It is used as a reference marker, or “diagnostic gold standard,” to which other breast cancer markers are compared. Low level of CA15-3 can be detected in saliva, and correlated with serum concentration closely. There are several advantages to detect CA15-3 in saliva such as: 1.Easy collection. 2. Repeatable without pain. 3. Reflect real time status of body status. There are also several difficulties still need to be overcome such as: 1.The variance of saliva detection in different collection methods. 2. The level of CA15-3 in saliva is up to 10-fold lower than that in serum. 3. To our knowledge, there are no commercially available salivary CA15-3 standards or controls to evaluate the performance of this detection method. In this study, we have to prepare varying concentrations of salivary CA15-3 samples at physiologically relevant concentrations.
4. It is unclear about processing variance for saliva CA15-3 detection. In this study, we prepared CA15-3 samples in saliva by matrix replacement method and analyzed intensity responses to the samples at various concentrations of CA15-3.in saliva by the Biacore SPR system and Au/ZnO thin film SPR system. The linear detection range with the SPR system based on thin-film Au/ZnO was 2.5–20 U/mL (the cut-off point in cancer patients is around 4 U/mL). The linear range with the Biacore SPR system was 40–300 U/mL. Previous reports from the Streckfus Lab have shown that the saliva CA15-3 concentration for healthy controls is about 3.19 ± 0.52 U/mL; for benign breast tumors, about 7.23 ± 1.58 U/mL; and for malignant breast cancer tumors, about 10.90 ± 3.44 U/mL. The Au/ZnO thin film SPR sensor system used in this study showed a linear response to CA15-3 across this entire concentration range.
The KD values for the Biacore and our Au/ZnO chip were calculated to be 381 and 17 U/mL, respectively. Obviously, the KD of the Biacore system was much higher than that of our Au/ZnO SPR chip. This difference is mainly due to the limitations of the dextran matrix to present the probe in a homogeneous conformation with its target. CM5 matrix has intrinsic limitations in terms of steric hindrance, heterogeneities in the density and conformation of probes inside the gel, and kinetic resistance due to improper molecular orientation and mass transfer that cause an apparent decrease in the reaction-rate constant. Thus, this newly developed thin film SPR system is expected to be quite useful in the clinical diagnosis of cancer progression.
These results show that thin-film Au/ZnO-based SPR systems have higher sensitivity and can be used for measuring the levels of CA15-3 in human saliva without concentrating the samples .

並列關鍵字

saliva carbohydrate antigen 15-3 ； surface plasmon resonance ； gold/zinc oxide ； Biacore SPR

參考文獻

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