微量生物分子檢測系統

Abstract

近日來生物分子檢測系統快速發展，並且大量應用在癌症的早期篩檢方面。常見的系統如immunoradiometric assay(IRMA)、fluorescence immunoassay(FIA)、chemiluminescence immunoassay(CLIA)、enzyme-linked immunoassay(ELISA)都頗有成效，但是在系統的靈敏性和專一性方面，仍然有許多改善空間。 本研究為了增加生物分子檢測的靈敏性，採用多壁奈米碳管當做基材，利用其高比表面積的特性，使系統能檢測的生物分子比一般固體基材更多，從而增加靈敏度。並且以抗體和抗原之間的專一性結合(specific binding)為基礎，讓系統中的抗體對生物分子或癌症抗原進行抓取。而被抓取的抗原，將會和系統中的偵測抗體-DNA片段複合物再做結合，其中DNA片段可用PCR技術複製，從而達到放大訊號，增加靈敏度的目的。 為了增加系統的專一性，而使微量生物分子檢測更加精確，本研究先在矽基板上以微波電漿輔助化學氣相沈積法(MPCVD)覆蓋一層疏水性的鑽石薄膜(diamond film)來減少親水性蛋白質與親水性基板之間的非專一性結合，然後在其上以熱燈絲化學氣相沈積法(HFCVD)成長出多壁奈米碳管(multiple-walled carbon nanotubes)。並將奈米碳管酸處理後接上聚氧乙烯二胺(PEG bis-amine)，利用其立體障礙在液體之中的泳動現象，以及帶負電而產生的斥力，來減少非專一性結合的發生。 實驗結果顯示，在氫氣流量為300 sccm，甲烷流量20 sccm，氮氣流量1.5 sccm，腔體壓力100 torr，微波功率1200 W，成長時間180分鐘，並且在580°C下做一小時的熱處理的鑽石膜，最能兼具疏水性與鑽石膜品質，並且便於後續奈米碳管生長。而氮氣流量140 sccm、乙炔流量17.5 sccm，成長溫度800°C，前趨物昇華溫度200°C，成長時間120分鐘，則能夠生長出比表面積大，粗細均勻，長度較長，且較具方向性的奈米碳管。並且由實驗結果得知，本研究建構的生物分子檢測系統其偵測極限約在0.02μg/ml，而非對應性的抗原，只要濃度不超過200μg/ml，就不會因為非專一性結合而產生偽陽性的訊號。

Biomolecule detection system has been rapidly developed, and it has also been generally applied onto early diagnosis of cancer. Commonly seen system such as immunoradiometric assay(IRMA)、fluorescence immunoassay(FIA)、chemiluminescence immunoassay(CLIA)、enzyme-linked immunoassay(ELISA)、immuno-polymerase chain reaction(Immuno-PCR) are all functioning effectively. Among them, there is a system which is called immuno-PCR is working through specific binding between antibody in the system and the antigen/biomplecules. The antigen which is captured will be combined with detection antibody –DNA fragment complex in the system, and the DNA fragment can be duplicated by PCR technology to enlarge the signal. Therefore, the sensitivity of immuno-PCR is much better than the commonly used ELISA system. So far we have known that immuno-PCR is to fix antibody on glass substrate by any means in order to catch antigen. However, the hydrophilic antigen or proteins are easily attached to hydrophilic glass substrate, and cause non-specific binding which leads to the inaccuracy in examination result. To improve the accuracy in examination result, a layer of hydrophobic diamond film has been grown on silicon substrate by MPCVD to decrease non-specific binding, subsequently the multi-walled carbon nanotubes are grown on it by HFCVD. After that the substrate processed an acid treatment, and then connected with PEG bis-amine, which can decrease non-specific binding by its steric repulsion, and the repulsion due to its negative electricity, which caused a waving effect . Moreover, it was connected to the C-terminal of α-1 antitrypsin antibody, and let the N-terminal of antibody can still remain active and caught the corresponded α-1 antitrypsin protein. After that we added in the detection antibody, that is, biotinylated α-1 antitrypsin antibody, which could be captured by the α-1 antitrypsin protein on the substrate. At the same time, the detection antibody was connected to the biotinylated DNA fragment through streptavidin, therefore we could amplify the signal by PCR.

In order to know the sensitivity and specificity of the system, α-1 antitrypsin proteins with different concentration have been detected. Polylysine has also been added in to compare that if the non-specific binding has diminished or not. The experiment result turns out that the diamond film which is under 300 sccm hydrogen, 20 sccm methane, 1.5 sccm nitrogen, 100 torr chamber pressure, 1200W microwave power, 180 minutes growing time and processed for 1 hour heat treatment under 580°C demonstrates best quality of hydrophobicity and is also better for following growth of carbon nanotubes, while under the condition of 140 sccm nitrogen, 17.5 sccm C2H2, growing temperature 800°C, precursor sublimed temperature 200°C, growing time 120 minutes can grow the carbon nanotubes which is longer, has larger specific surface area, with uniform thickness, and the directionality. Also, from the experiment result, it shows that the detection limit of the trace biomolecule detection system built up by this research is about 0.02 μg/ml, and the unwanted signal will not appear if the concentration of non-specific protein is less than 100 μg/ml.