利用比例螢光法設計核酸感測器以用來偵測心血管因子有關之凝血酶

A ratiometric optical aptamer sensor for the detection of cardiovascular disease-associated thrombin

張嘉郝(CHIA-HAO CHANG)

指導教授 ： 周芳如

中原大學/理學院/化學系/碩士(2022年)

https://doi.org/10.6840/cycu202201637

摘要

本篇研究利用以核酸序列作為模板，合成之銀奈米團簇訊號探針(AgNCs probe)，並輔以磁性奈米粒子辨識目標物凝血酶，使其做為心血管疾病以及動脈粥狀硬化之檢測方法。該合成之銀奈米團簇訊號探針(AgNCs probe)，利用分別設計不同核酸模版序列，以及在不同合成環境及濃度進行一系列之優化，而可產出在波長557與609 奈米處有螢光產生，並藉由互補後探針構型之改變而產生比例螢光變化。首先是以凝血酶特異性核酸適體(Thrombin aptamer)改質之磁性奈米粒子為探針，分別以兩種策略進行凝血酶的檢測。第一種為藉由該適體事先與保護子序列(Protector DNA)進行互補，在有凝血酶存在時，使該保護子序列釋出並與銀奈米團簇核酸探針(AgNCs probe)互補後，可以開啟髮夾型輔助核酸(Helper DNA)，依序交替進行催化髮夾開啟及組裝，使得銀奈米團簇訊號探針在互補反應前後有螢光差異並且放大該差異值，該方法能夠檢測低於人體內凝血酶含量的human α-thrombin (0.05 mg/ mL)，並且與未含有凝血酶的組別有顯著差異(p = 0.0055)，以作為凝血酶的定量工具。另一種方式為凝血酶專一性適體與取代核酸(Displacement DNA)預先組成雙股核酸探針，在有凝血酶的條件下，該取代子核酸(Displacement DNA)被釋放，並進一步以立足點取代反應(Toehold-mediated strand displacement reaction)將核酸模板銀奈米團簇上的預先互補序列之保護子核酸(Protector DNA)進行置換。藉由螢光訊號下降的趨勢以建立與凝血酶的濃度相關性。目標為綜合以上兩種策略並調整選用的適體，該系統便具有潛力能針對其他生物標記物能夠選擇性的偵測。

關鍵字

適體 ； 銀奈米團簇 ； 心血管疾病 ； 凝血? ； 比例螢光

並列摘要

Cardiovascular disease (CVD) has been the leading cause of death in the world, mostly due to heart attack and stroke. It’s therefore of importance to detect CVD as early as possible; however, the early detection remains challenge because the regular examination is usually time-consuming and requires sophisticated instruments. In this study, we aim to develop an optical aptamer sensor for the detection of thrombin in blood sample. We use two kind of strategies. Upon the presence of the target, a pre-caged protector sequence is released from an aptamer/protector duplex, and the protector sequence opens up a hairpin probe with fluorescent silver nanocluster labeled. The fluorescence can be further amplified with the addition of a helper sequence by catalytic hairpin amplification. Such fluorescence change leads to a ratiometric signal that can be used to quantify the amount of the target. The other strategy is to employ a “turn-off” strategy by forming a fluorescent probe/protector sequence first, and the protector sequence is displaced by a displacement sequence that is pre-hybridized with the aptamer upon the addition of the target.

並列關鍵字

aptamer ； silver nanocluster ； cardiovascular disease ； thrombin ； ratiometric fluorescence

參考文獻

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