- 學位論文
矽晶片表面披覆奈米碳管結合免疫聚合酶連鎖反應應用於乳癌復發早期診斷之研究
Application of Carbon Nanotubes Coated on Silicon Wafer Combined with Immuno-PCR on the Early Diagnosis of Breast Cancer Recurrence
摘要
乳癌為台灣女性人口罹患率最高的癌症，若乳癌復發，有相當大的機率出現遠端轉移，造成無法治癒且預後較差的嚴重後果。現有的數種診斷方式，由於影像技術限制而不易發現腫瘤，且大部分的影像技術需要放射線，可能對病人的健康造成危害。因此，本研究利用免疫聚合酶連鎖反應作為診斷工具，利用抗原-抗體間的特異性反應，以及聚合酶連鎖反應能大量複製DNA的特性，架構一套兼具特異性和高靈敏度的檢驗方法，希望能由病人血清早期診斷乳癌復發，進而能夠及早治療，並延長患者的存活時間。 首先使用經氫氟酸處理的矽晶片為基材，成長簇狀奈米碳管，接著利用硝酸與硫酸混合溶液進行表面改質，使奈米碳管末端具有羧基，再接枝上聚氧乙烯二胺，最後將一次抗體(anti human CA 15-3 antibody)的C端固定在表面，並以水接觸角、掃描式電子顯微鏡、熱分析儀、傅立葉轉換紅外線光譜儀、拉曼散射光譜儀與十二烷基磺酸鈉-聚丙烯醯胺膠體電泳作材料修飾的評估分析。在免疫聚合酶連鎖反應的部分，首先以基材表面所固定的一次抗體捕捉乳癌抗原(human CA 15-3)，再與生物素標記二次抗體結合，最後以親合素架橋將生物素標記二次抗體和生物素標記DNA連接在一起，以引子將生物素標記DNA大量擴增，產物利用瓊脂膠體電泳以及微量分光光譜儀作分析判讀。 由實驗結果得知，在疏水性矽晶片表面成長簇狀奈米碳管，並接枝聚氧乙烯二胺，能夠有效減少蛋白質與DNA的非專一性吸附，降低免疫分析時的背景值。在本研究所使用的實驗參數下，免疫聚合酶連鎖反應對於CA 15-3的偵測靈敏度較酵素連結免疫吸附法約高出500-5000倍。
並列摘要
Breast cancer is the most commonly diagnosed cancer for women population in Taiwan. If breast cancer recurrence happens, there would be very high possibility for distant metastasis, which could not be cured and lead to a worse prognosis. Breast cancer is commonly diagnosed by breast images, which is not easy to detect the tumor due to technical limitations. Also, it accompanies with radioactive rays and may hazard patients’ health. In this study, we utilized immuno-PCR as a tool for diagnostics. Our purpose was to develop a precise (due to the specificity between antigen and antibody) and sensitive (due to the amplification of DNA by polymerase chain reaction) method to diagnose breast cancer recurrence from patients’ serum. Furthermore, we hoped that patients could be treated properly as early as possible for longer survival periods. First, silicon wafer, being treated by hydrofluoric acid, was used as the substrate, with bundled carbon nanotubes (CNT) coated on it. Then, we used HNO3 / H2SO4 solution to do the surface modification and carboxyl groups would be grafted on the tips of CNT. Next, polyoxyethylene bis-amine (PEG bis-amine) would be grafted and capture antibodies (anti human CA 15-3 antibody) were conjugated. Water contact angle measurements, scanning electron microscope (SEM), thermal analysis (TA), Fourier transform infrared spectrometer (FTIR), Raman scattering spectrometer and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) would be used to prove that the surface modification was successful. The capture antibodies conjugated on the CNT were used to detect antigens (CA 15-3). Biotinylated detection antibodies (secondary antibodies) were further bound to the antigens. Free streptavidin was used to link biotinylated DNA to the biotinylated detection antibodies. The biotinylated DNA was amplified by PCR, and analyzed by agarose gel electrophoresis and NanoDrop. According to the experimental results, the substrate, PEG bis-amine grafted CNT coated on the hydrophobic surface of silicon wafer, could prevent non-specific binding of protein and DNA and then reduce background noise in immunoassays. By using the parameters in this study, the sensitivity for carbohydrate antigen 15-3 (CA 15-3) detection by using our immuno-PCR system was 500-5000 folds higher than ELISA.