薄膜側向流於多孔聚合物表面對生物性樣品之感測

Membrane-Based Lateral-Flow Detection of Biological Samples on Porous Polymer Surfaces

傅佳瑋(Chia-Wei Fu)

指導教授 ： 吳瑞璋

中原大學/工學院/化學工程研究所/碩士(2014年)

https://doi.org/10.6840/cycu201400532

摘要

本研究利用抗體─抗原免疫反應以及DNA序列上特殊修飾標的物，進行薄膜側向流偵測，並以抗體接上膠體金顯色進行判讀。
研究第一部分使用三維孔洞具生物活性環氧官能基氣凝膠(AG60-GL10)，在最佳化條件37℃、4小時及90% 相對溼度下，使用goat anti -mouse IgG抗體鋪為試條上的測試線，並鋪中孔洞二氧化矽(AG60)作為試條基材。在pH=10.5，mouse IgG模擬抗原濃度大於30µg/ml時，膠體金顆粒與目標抗原具有最佳的結合效果。由UV-vis吸收光譜及TEM驗證，已將mouse IgG成功接上膠體金。薄膜側向流偵測其肉眼能觀測之訊號強度所需濃度為0.16 µg，成功將新穎材料氣凝膠與生物性免疫感測試條結合。
研究第二部分利用DNA間序列特殊修飾標的物做專一性偵測，各別偵測禽流感(Avian Influenza) H5N1中的H5序列標的物及HLA-B*5801標的物。在pH= 8.5，mouse anti-dig抗體濃度應大於10µg/ml時，與膠體金顆粒達最佳的結合效果。由UV-vis吸收光譜及TEM驗證，已將抗體成功接上膠體金。結果發現H5標的物雙股核苷酸於試條的兩端施加直流電壓12V時，與未加電壓時的偵測極限皆為2ng，證實標的物雙股核苷酸並不適用於施加電場之方法來提升訊號。以H5作未純化HLA-B*5801之對照組，發現HLA-B*5801樣品之序列長度為320bp，且樣品量以20µl呈現訊號效果最佳。而純化後之HLA-B*5801其肉眼所能觀測之訊號強度之最低偵測濃度為2ng，薄膜側向流為敏感高效率的快速偵測。

關鍵字

硝化纖維膜 ； 薄膜側向流 ； 氣凝膠 ； 核苷酸 ； 直流電

並列摘要

In this study, biological samples, either sequence-specified DNA segments or protein, were detected on porous polymer surfaces incorporated with an immune reaction and color development by nano-size gold particles. There were two parts in this thesis:
Part I: the epoxy-modified bioactive mesoporouse aerogel(AG60-GL10) was spread on a sticky plastic card to become a base of lateral-flow membrane layer. The goat anti-mouse IgG antibody was then printed on the aerogel as the detection line for detecting its corresponding antigen. The antibody was immobilized on the aerogel at the optimal condition of 37 ℃ for 4 hours with the relative humidity of 90%. The labeling condition of nano-gold particles onto the antibody was determined as pH=10.5 and antibody concentration at least 35µg/ml, to avoid the complex of gold-antibody from aggregation. This labeling process was validated with the UV-vis spectrum and transmission electron microscopy (TEM). The lowest detection limit of the biological sample was determined as 0.16 µg by unaided eyes on a novel bioactive aerogel material.
Part II: The Avian-Influenza (AI) H5 DNA and human leukocyte antigen B*5801 DNA’s were individually detected on the membrane-based later-flow strips. The optimal conjugation condition of mouse anti-digoxin with colloidal-gold particles was determined as pH=8.5 and antibody concentration at least 10µg/ml, to avoid the complex of gold-antibody from aggregation. This labeling process was also validated with the UV-vis spectrum and transmission electron microscopy (TEM). Application of 12 volts on the membrane-based later-flow strips received an identical result for either double or single-stranded DNS’s. HLA-B*5801 was determined as a 320-bp DNA segment and had the detection limit of 2 ng, same as AI H5 samples.

並列關鍵字

DNA ； Silica ； membranes-based ； lateral-flow ； colloidal gold

參考文獻

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