標題: 可偵測低濃度生物標誌之數位微流體免疫檢測晶片
Development of digital microfluidic immunochip for detecting low level of biomarkers
作者: 黃正鄴
徐文祥
Huang, Cheng-Yeh
Hsu, Wensyang
機械工程系所
關鍵字: 微流體;免疫檢測;microfluidics;immunoassay
公開日期: 2016
摘要: 本研究旨在研發一僅需極少量樣品體積,即可偵測低濃度生物標誌之數位微流體免疫檢測晶片,包含開發兩項新型數位微流體操作技術,應用於免疫檢測分析晶片。第一項技術,在完成磁珠式免疫檢測分析後,將磁珠進行聚集,可不需更動原有免疫檢測分析流程,即達到增強偵測訊號並提升靈敏度。透過系統化的研究磁珠聚集帶來的訊號增強效果,發現只有在適當的雙板間距下,磁珠聚集才能穩定地增強訊號。本研究並以第一型腫瘤壞死因子受體(sTNF-RI)為免疫檢測分析模型,配合傳統磁珠式免疫檢測分析流程,成功證實此偵測方式可降低偵測極限至15 pg/mL,並減少變異係數至3%。第二項技術,以一連串不同大小的電極達成少顆數磁珠操控,及高效率的清洗,且在清洗過程中完全不會漏損磁珠。實驗結果顯示,在磁珠數100顆以內,僅需要一次清洗步驟即可達到98%的清洗效率,且無任何磁珠漏損。在過去的文獻中,已經證實減少磁珠可以增加每顆磁珠表面的鍵結量,進而有效的提升偵測訊號。因此,本研究進一步整合這兩項技術,設計一新型數位微流體免疫檢測晶片,以少量磁珠進行磁珠式免疫檢測分析,目的是快速且準確的檢測低濃度生物標誌。本研究同樣以第一型腫瘤壞死因子受體為免疫檢測分析模型,進行比較,偵測極限可降低至3.14 pg/mL,樣品需求量減少至200 nL,整個免疫檢測分析可在一小時內完成。結果成功驗證,此數位微流體免疫檢測晶片樣品需求量少,偵測極限低,穩定度高。此一免疫檢測晶片未來可有助於不同臨床疾病的診斷及疾病生物學上的研究,尤其在試管嬰兒的胚胎培養中,所使用的胚胎培養液體積相當小,僅5-10 μL,且生物標誌濃度相當低,少於數十pg/mL,難以傳統免疫檢測進行分析。未來此一免疫檢測晶片將有助於監測及探討胚胎培養過程中不同胚胎因子濃度變化,希望能輔助目前僅能以外觀等級判定生長狀態的胚胎挑選,找出較佳的植入胚胎,進一步減少植入胚胎數,並提升懷孕率及分娩率。
This thesis describes the development of a digital microfluidic immunochip for detecting low level of biomarkers in an ultra-low volume sample. Two new techniques on a digital microfluidic (DMF) setup are developed for performing immunoassays. The first technique enhances the detection signal and sensitivity even at the post-assay stage without altering the original assay procedures chemically or biologically by employing magnetically triggered post-assay aggregation of beads in a DMF setup. The effect of bead aggregation in such an immunoassay system is systematically investigated. Bead aggregation is shown to stably enhance the fluorescence intensity in DMF device with a proper gap distance. In our soluble tumour necrosis factor receptor I (sTNF-RI) model immunoassay, limit of detection (LOD) 15 pg/mL and coefficient of variation (CV) 3% are achieved. The second technique enables the manipulation of low number of beads to provide stronger fluorescence signal per bead with high washing efficiency by utilizing a series of unequal electrodes. The result shows that only one wash step is required to achieve 98% washing rate without any bead loss at bead number less than 100 in a droplet. Decreasing the starting number of antibody coupled beads in immunoassay is shown to effectively elevate the responding signal, since less starting bead number would allow more antigens to bind to the surface of each bead. Therefore, by combining both techniques, our sTNF-RI model DMF immunoassay with LOD 3.14 pg/mL is achieved, and only 200 nL sample volume and less than 1 hour of analysis time are needed. This DMF immunoassay platform hence allows a small consumption of sample volume at a nanoliter level to achieve low limit of detection at the pg/mL level. Another aspect demonstrated is a decreased uncertainty. It may aid in some clinical diagnostics by providing modified assays with lower required sample and LODs to give insights into disease biology. Especially for in vitro fertilization (IVF), the sample quantity of embryo culture medium (5-10 μL) and concentration of biomarkers (below several tens of pg/mL) are all very low, which is hard to use conventional immunoassay to detect. The developed DMF immunoassay can act as a powerful embryo immuno-detection chip to detect and investigate different embryonic growth factors during embryo culture in IVF. It is hoped that the proposed embryo immuno-detection chip can provide quantitative evaluation of cultured embryos to alter the current morphology-based embryo selection, and reduce the implanted embryo number with good pregnancy rate and delivery rate.
URI: http://etd.lib.nctu.edu.tw/cdrfb3/record/nctu/#GT079914801
http://hdl.handle.net/11536/139862
Appears in Collections:Thesis