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研究生:楊景雯
研究生(外文):Yang, Ching-Wen
論文名稱:石英晶體微天平的脂多醣體檢測
論文名稱(外文):Determination of lipopolysaccharide based on Quartz crystal microbalance system
指導教授:張憲彰
指導教授(外文):Fong-Chih Su
學位類別:碩士
校院名稱:國立成功大學
系所名稱:醫學工程學系
學門:工程學門
學類:綜合工程學類
論文種類:學術論文
論文出版年:1997
畢業學年度:86
語文別:中文
論文頁數:75
中文關鍵詞:石英晶體微天平生物感測器抗生素脂多醣體白蛋白去除
外文關鍵詞:Quartz crystal microbalanceBiosensorAntibioticsLipopolysaccharideDeprotein
相關次數:
  • 被引用被引用:2
  • 點閱點閱:241
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  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:1
脂多醣體(Lipopolysaccharide﹐LPS)為存在於革蘭式陰性菌細胞壁外膜
上之特有物,在人體中為一種內毒素,會引發敗血症、休克等現象。傳統
上檢測LPS以Limulus amebocyte lysate (LAL)測試法為最普遍﹐其利用
鱟血液中的阿米巴細胞(Amebocyte)會與含有LPS檢品產生僅憑肉眼即可視
之凝膠﹐然此法費用高昂且屬半定量式﹐基於此,本研究改以靈敏性高之
石英晶體微天平(Quartz crystal microbalance,QCM)系統為感測器之換
能元件(Transducer),於其上設計予可與LPS相互作用而形成鍵結,期能
構成一價廉且更高靈敏度之感測器。在QCM之金電極表面上固定抗生素
PolymyxinB (PmB)為生物感測元件,藉之與待測物LPS結合造成QCM電極的
負載更形增加,而導致頻率下降以檢測LPS含量。將疏水性之金電極浸漬
於含0.05 M 4,4''-ditholdi(n-butyric acid)(DTBA)之乙醇,而使電極表
面改質形成帶有COOH官能基,其次再經由1-ethyl-3-(3-dimethyl-
amionpropyl) carbodiimde (EDC)及N-hydrosuccinimide (NHS)的活性化
後,將帶一級胺基的PmB固定於電極表面上。以此製備的LPS感測器具高靈
敏性,其最低之檢測極限濃度為25 pg/ml。修飾PmB之石英晶體微天平可
偵測LPS含量,歸因於LPS結構中疏水性之lipid A會與PmB之疏水區結合,
及LPS帶負電之脂肪酸鏈與帶正電之 PmB產生靜電吸引力相結合所導致。
此外電極於使用後可經pH為2.5的Glycine-HCl緩衝液充份水洗後重覆使用
,其線性範圍雖有下降之趨勢,仍可維持2.5 ng/ml ~ 22.5 ng/ml。本檢
測系統亦應用於含牛血清白蛋白(BSA)的樣品,發現BSA會直接吸附於感測
器表面上,而形成檢測上極大的干擾。為此,利用ZnSO4及
Trichloroacetic acid (TCA)兩法進行去除BSA的處理,其後並以Lowry法
作為殘留蛋白量之評估。結果顯示,以ZnSO4法可有效地去除BSA,並對
LPS含量之檢測影響較TCA法小,故可以ZnSO4處理運用於血清檢品之偵測

Lipopolysaccharide (LPS) an endotoxin which existed in the
outer membrane of cell wall of Gram negative bacteria G(-) could
induce some diseases such as sepsis, endotoxin shock, and so on.
Limulus amebocyte lysate (LAL) test is a conventional method, it
was performed by gel-clotting between LPS and ambeocyte.
However,this method defeats in high cost and semi-
quantification,Quartz crystal microbalance (QCM), was developed
to be a less cost and highly sensitive transducer. Adsorbing the
analyte on the quartz surface, it would cause a proportionally
decreasingon resonance frequency of oscillation. In this study,
we used an antibiotics, polymyxin B (PmB), as a sensing
elementwhich was coupled on the surface of QCM that
couldintroduce a new function for sensing LPS and show the shift
ofoscillation frequency. PmB immobilized on the surface of QCM
was via coupling method, immersing the gold-deposited crystal
into the ethanol solution containing 4,4''-dithiodi-(n-
butyricacid) (DTBA) for 2 hr at room temperature. After washing,
the treated crystal was activated with coupling agent,
1-ethyl-3-(3-dimethyl-amionpropyl) carbodiimine(EDC) and N-
hydrosuccinimide (NHS). Subsequently, the crystalwas immered
into aqueous solution containing 2 mg/ml of PmB. The resulting
sensor showed a linear range of LPS concentration between 2.5
ng/ml and 22.5 ng/ml and allow limit detection as 25 pg/ml.
However, we found such a PmB modified sensor would be interfered
by the adsorptionof bovine serum albumin (BSA). To extend a
method to the clinical application, an available deprotein
treatment was necessary. We discussed two deprotein agents,
ZnSO4 and trichloroacetic acid (TCA). As a result, the LPS
sample mixed with BSA was treatedZnSO4 that mainly precipitated
BSA showed a higher effect thanTCA method.Keyword: Quartz
crystal microbalance (QCM), Biosensor, Antibiotics,
Lipopolysaccharide, Deprotein
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