發展表面電漿共振生物感測器於藥物篩選流程之應用－以CYP2D6抑制試驗為例

Development of SPR Biosensor Apply to the Drug Screening Process - Focus on CYP2D6 Inhibition Assay

林勁戎(Jing-Rung Lin)

指導教授 ： 林啟萬

國立臺灣大學/電機資訊學院/電機工程學研究所/碩士(2006年)

https://doi.org/10.6342/NTU.2006.01836

摘要

表面電漿共振生物感測器擁有偵測時免標記以及具備高靈敏度的優點。本研究利用此特色，針對藥物開發過程中所需的高通量篩檢，發展表面電漿共振生物感測器應用於CYP450抑制試驗，希望能減少篩檢流程，進而縮短試驗時間，達到加速篩檢的目標。
相較於一般以螢光測量的方式必須添加螢光標記，使用表面電漿共振生物感測器可省去標記的步驟，加快檢測速度。CYP2D6是CYP450中的一個重要的酵素，除了負責代謝大量的臨床藥物外，還有因個人或種族而異的多樣性變化。因此在此以CYP2D6為例，測量50%抑制濃度來比較非抑制性藥物和抑制性藥物的差別。實驗中固定debrisoquine感測器表面，當作探針用以偵測CYP2D6的活性。由於在量測過程中，表面感測區的非特異性吸附現象會對量測結果造成少許的誤差，所以實驗中也量化非特異性吸附的誤差大小，再從結果中扣除校正。酵素和藥物探針間的結合反應和酵素活性強弱的關係也可由活性和非活性的量測結果當中大略觀察出來。兩種選擇用以測試的藥物分別是非抑制性的dextromethorphan和抑制性的quinidine，針對CYP2D6的50%抑制濃度量測結果分別是1.3456 μM和0.32129 nM，由此實驗結果可以看出待測藥物的濃度和酵素及探針的結合反應有一定程度的相關，且藥物因抑制性的差別而有不同的50%抑制濃度。兩種藥物dextromethorphan和quinidine校正後50%抑制濃度分別為0.8624 μM和0.07814 nM，皆較校正前小，而依舊可以看出兩者在抑制能力的差異。本研究結果顯示表面電漿共振生物感測器可以透過表面固定適當的藥物探針而有機會應用於CYP450抑制試驗，且量測50%抑制濃度的結果可以看出抑制藥物和非抑制藥物的差別，雖有無法避免的誤差，但可以藉由修正改善。
以本研究的實驗結果為基礎，結合自動化的應用，未來可利用多通道的流道設計，將表面電漿共振生物感測器開發成具有高通量能力的藥物篩檢工具。

關鍵字

表面電漿共振生物感測器 ； CYP450 ； 50%抑制濃度

並列摘要

The advantage of surface plasmon resonance (SPR) biosensor includes label-free and high sensitivity. Due to the characteristics of SPR biosensor and the requirement of high throughput screening (HTS) in drug development, the aim of this study is to develop SPR biosensor for rapid screening in CYP450 inhibition assay.
Compared with the common fluorescence-based method, SPR biosensor can neglect the label step in procedure and accelerate the assay speed. CYP2D6 is an important isoenzyme responsible for a large part of marked drugs. Pharmacogenetic Polymorphism is also observed in CYP2D6. Here the measurement of 50% inhibitory concentration (IC50) for CYP2D6 with non-inhibitor (dextromethorphan) and inhibitor (quinidine) using SPR biosensor is demonstrated. The probe drug, debrisoquine, is immobilized on sensor surface to detect the activity of CYP2D6. Nonspecific binding can lead to measurement error. Here the magnitude of error is also measured and the results are corrected, and the correlation between enzyme activity and binding reaction is also observed. Results of IC50 measurement are 1.3456 μM and 0.32129 nM for dextromethorphan and quinidine. After correction, IC50 of dextromethorphan and quinidine are 0.8624 μM and 0.07814 nM, both are smaller than uncorrected value. It shows that testing substrate concentration is associated with binding reaction and enzyme activity. This study represents that SPR biosensor can be considered as a tool for CYP450 inhibition assay after the selected probe drugs are immobilized on sensor surface. These results show the difference between the non-inhibitor and the inhibitior. From the results of IC50, the distinction between non-inhibitor and inhibitor are observed. The unavoidable error can be estimated and used for correcting IC50 measurement results.
Because the multi-channel design and automation can be carried out in SPR biosensor, the results of this study suggest that biosensor based on SPR has the potential to provide a HTS tool for drug screening in the future.

並列關鍵字

SPR biosensor ； CYP450 ； IC50

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