建立磁珠搭配適體感測平台應用於快速且選擇性地螢光定量分析耐甲氧西林金黃色葡萄球菌

Magnetic beads combined with aptasensor for fast and selectively fluorescent quantification of methicillin-resistant Staphylococcus aureus

蔡明璋(Ming-Chang Tsai)

指導教授 ： 王俊棋

高雄醫學大學/藥學院/藥學系碩士班/碩士(2020年)

摘要

本篇研究係以適體（aptamer）搭配奈米磁珠以及螢光探針組成之生物傳感器，用以針對methicillin 抗性之金黃色葡萄球菌（methicillin-resistant Staphylococcus aureus, MRSA）進行檢測。MRSA的生成來自於過度使用抗生素治療金黃色葡萄球菌，因而導致變異並產生抗性，由於MRSA易於傳播且難以治療，因此除了研發有效之抗生素外，檢測MRSA的技術開發也逐漸興起，並用以作為病症上的判斷或是預防醫療等用途。
本研究以鏈黴親和素塗層之磁珠（streptavidin magnetic beads, SMBs）作為aptamer之載體，透過0.2 µM aptamer與MRSA特異性結合後，加入20 µL磁珠以結合游離之aptamer，並以磁鐵將磁珠沉澱之後，加入0.015 µM螢光探針，並以400 mM氯化鈉溶液輔助螢光探針與aptamer序列互補結合穩定，再利用磁鐵沉澱磁珠，並取上清液進行螢光測定，透過螢光強度的高低來對應檢測之細菌濃度。
結果證實所開發檢測方法具有簡單操作、快速檢測並能特異性辨識MRSA，且檢測極限低至860 cfu/mL，並且能應用於生物檢體，如尿液中進行檢測。

關鍵字

適體 ； 耐甲氧西林金黃色葡萄球菌 ； 生物傳感器

並列摘要

In this study, a biosensor composed of aptamer, nanomagnetic beads, and fluorescent probes were used to detect methicillin-resistant Staphylococcus aureus (MRSA). The generation of MRSA comes from the excessive use of antibiotics to treat Staphylococcus aureus, which leads to mutations and resistance. Because MRSA is easy to spread and difficult to treat, in addition to the development of effective antibiotics, the development of technologies to detect MRSA has also gradually emerged and used it as a judgment on disease or preventive medical use.
In this study, streptavidin magnetic beads (SMBs) coated with streptavidin were used as the carrier of aptamer. After 0.2 µM aptamer was specifically combined with MRSA, 20 µL magnetic beads were added to bind the free aptamer, and the magnet After the magnetic beads are precipitated, add 0.015 µM fluorescent probe, and use 400 mM sodium chloride solution to assist the complementary binding of the fluorescent probe to the aptamer sequence to stabilize, and then use the magnet to precipitate the magnetic beads, and take the supernatant for fluorescence measurement. Corresponding to the detected bacterial concentration through the intensity of fluorescence.
The results confirmed that the developed detection method has simple operation, rapid detection and accurate identification of MRSA, and the detection limit is as low as 860 cfu/mL, and can be detected in biological samples, such as urine.

並列關鍵字

aptamer ； methicillin-resistant Staphylococcus aureus ； biosensor

參考文獻

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