紅外線吸收光譜與拉曼光譜作為酵素免疫分析儀之可行性研究

Feasibility Study on FTIR Spectroscopy and Raman Spectroscopy as the Reader of Enzyme-Linked Immunosorbent Assay

蔡承諺(Cheng-Yen Tsai)

指導教授 ： 江建文

國立臺灣大學/理學院/化學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201700950

摘要

酵素免疫分析法(Enzyme-Linked Immunosorbent Assay , ELISA)是現今相當重要的體外診斷技術，因為癌症的治癒率與接受治療的早晚有密切關係，為了提早發現，人們會定期至醫院做篩檢，因此體外診斷的需求逐年升高，在2016年的免疫檢測市場規模達到171億美元。在卵巢癌的檢測上，血紅素結合蛋白被作為癌症前置因子來判定患者罹癌之潛在風險，針對此蛋白質定量的酵素免疫分析法也已問世並採用。當此蛋白質在血液的濃度過高，表示有很高的風險罹癌，而在此蛋白質過少時，表示血液中溶血症狀嚴重。所以我們認為血紅素結合蛋白濃度的測量是重要的，目前的酵素免疫分析法主要是以UV-vis做為分析儀，並以比色法方式定量血紅素結合蛋白，而TMB2+這個化合物的生成，是顯色深淺的主因。不過，此法的偵測濃度範圍並不大。因此，我們嘗試以紅外線吸收光譜及表面增強拉曼光譜作為分析儀，改善現有酵素免疫分析法。在紅外線吸收光譜中，我們將各個標準檢液滴在二氟化鈣鹽片上，以真空法除去溶劑，並測得紅外線吸收光譜，我們可以看到TMB2+在1572 cm-1之吸收峰。而在表面增強拉曼光譜中，我們將標準檢液與金奈米粒溶液混合，而金奈米粒溶液可以增強TMB2+的訊號，因此測得1605cm-1的峰值，而這個峰值的強度會隨著TMB2+濃度上升而增加。根據以上，我們展示了FTIR與SERS作為ELISA分析儀的可行性，並增加此法可偵測的濃度範圍，我們非常期待這個新方法能夠使現今的ELISA技術更好，為醫療做出貢獻。

關鍵字

酵素免疫分析法 ； 紅外線光譜 ； 拉曼光譜

並列摘要

Enzyme-Linked Immunosorbent Assay (ELISA) is a very important in vitro diagnostic device (IVDs). Because the early treatment is able to improve the survival rate of cancer patients, where they could go to hospital for screening. Thus, the demand of in vitro diagnosis is increasing annually. In 2016, the global market of immunoassay reached USD 17.16 billion. In ovarian cancer, haptoglobin have been used as a prognosis factor to evaluate the risk of cancer, and we can find clinical tests based on haptoglobin ELISA kits. When the concentration of this protein in the blood is high, it means that increased risk of having ovarian cancer. In contrast, excessively low levels of haptoglobin indicates serious hemolysis. We believe that it is important to estimate the concentration of haptoglobin. However, The detection range of current ELISA reader is poor. Thus, we used FTIR spectroscopy and surface enhance Raman spectroscopy (SERS) as the readout of ELISA to improve the detection range of ELISA. In FTIR based ELISA, we dropped each standard sample on CaF2 window and the solvent was removed by vaccum. Subsequently, the samples was scanned by FTIR. It showed that a peak at 1572cm-1, which was the absorption of TMB2+, a product reacted by chromogen substrate(TMB) and HRP. In SERS, the sample was mixed with gold nanoparticles, as gold nanoparticle could enhance the signal of TMB2+. A peak at 1605 cm-1 was detected. It found that the intensity of the peak was increasing as the concentration of TMB2+ increased. In conclusion, we demonstrated the feasibility that FTIR and SERS could be used as ELISA readout, and the new method also expands the detection range of Hp. We look forward to improving existing ELISA methods by this research.

並列關鍵字

ELISA ； FTIR ； Raman

參考文獻

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