電漿和接枝聚合處理對QCM表面固定DNA的影響及病源菌檢測之研究

The Study of Immobilization of DNA on Plasma and Graft Polymerization Treated QCM Surface for Food Pathogens Detective

詹婉禎(Wan-Chen Chan)

指導教授 ： 陳克紹

大同大學/工程學院/材料工程學系所/碩士(2004年)

摘要

石英晶體振盪器(quartz crystal microbalance，QCM)是屬於超微量質量感測器之一，其振盪頻率會受表面吸附質量的改變而影響。然而為使其能更具廣泛應用性並且具可逆性，故可於其無機表面鍵結一層可辨識生物分子的物質，使其吸附生物分子時造成相對的頻率變化，此原理即可用於生物感測器之製作，例如可藉由像核酸雜交或抗原-抗體間的交互作用，以偵測因專一性結合所引起的質量改變，其同時具有選擇性、靈敏性和允傍漭X使用的可攜性等優點。本研究之目的是利用電漿能均勻聚合沉積膜薄，以及在無機基材上附著性優良等特點，使無機基材表面有機化，以作為無機物與有機分子的介面層，且可產生過氧化基或活化點，再以UV光接枝聚合AAm，而將胺基固定在QCM上，再經過一些架橋劑及增加胺基的單體溶液處理後，使QCM表面上可使用戊二醛來固定經過設計的含單股胺基DNA (ssDNA) 片段，隨後即可用來檢測被PCR擴增的目標DNA，從振盪頻率的改變來確認目標物之量。主要探討電漿沉積HMDSZ和接枝聚合親水性單體AAm對探針固定量及雜交量之影響。結果證實利用電漿沉積 (HMDSZ 50 mtorr、1min、20W) 和接枝聚合 (AAm 10 wt%、1000W、20min) 法確實可將設計的核酸探針 (大腸桿菌O157:H7的主要致病基因: eaeA基因序列)固定於QCM上(頻率下降量=130~170 Hz)，並可進行大腸桿菌O157:H7的檢測 (頻率下降量=220~270 Hz)。

關鍵字

生物分子固定 ； 病源菌檢測 ； 表面接枝 ； 電漿改質 ； 石英晶體振盪器

並列摘要

Quartz crystal microbalances (QCMs) are transducers for chemical and biochemical sensing in general. The oscillation frequency of QCM will be affected when the adsorbed mass on the surface changed, so in view of this characteristic, it can be used widely to all kinds of mass sensor applications. If immobilized the NH2-biomolecule on QCM surface. Such as single strand DNA, then from the frequency shift measured can detect DNA hybridization reaction. In this study, the HMDSZ plasma is utilized to deposit organic silicon thin film on the surface of QCM in order to make an interface layer between the inorganic material and organic molecule. Consequently, the surface UV-light grafting polymerization of AAm can provide useful functional sites for binding enzyme or the DNA fragment of bacillus. Hence, GA as cross-linking reagent to chemical bonding NH2-oligonucleotide probes on the surface of QCM. Experimental results indicated that combination of plasma deposition (HMDSZ 50 mtorr、1min、20W) and graft polymerization (AAm 10 wt%、1000W、20min); the biomolecul with –NH2 groups (such as DNA probe) can be immobilization effectively on QCM surface. And it has good detection sensitivity for the pathogenic gene eaeA of Escherichia coli O157:H7 than other control experiments; meanwhile the fabricated DNA-QCM sensor could differentiate from the other treated bacteria.

並列關鍵字

DNA immobilization ； Food pathogens detection ； Plasma polymerization ； QCM ； surface graft polymerization

參考文獻

Biosens. Bioelectron. 14, 443-456.

modified enzyme in an enzyme switch response to glucose. Anal. Chem. 72,

multisite microelectrodes for detection and elimination of interferences

electrodes immobilized on gold using self-assembled layers. Anal. Chem.

determination of glucose.Anal. Chim. Acta 363, 215-220.