第一部分.利用量子點與奈米碳球研發免疫電化學感測器以偵測腫瘤標幟蛋白質分子 ; 第二部分.研發利用雙重乳化法與針式過濾器合成微脂體之裝置

Part I. Carbon Nanoparticle-Enhanced Immunoelectrochemical Detection for Protein Tumor Marker with Cadmium Sulfide Biotracers ; Part II. Synthesis of Liposomes Using Double Emulsion Template and Syringe Filter

林業鈞(Yeh-Chun Lin)

指導教授 ： 何佳安

國立清華大學/理學院/化學系/碩士(2009年)

摘要

本論文的第一部分為研發偵測腫瘤標幟蛋白質分子之免疫電化學感測器。研究中透過修飾奈米碳球、聚乙烯亞胺與抗體於網版印刷電極，研發出一套靈敏的免疫電化學系統，可用於偵測腫瘤標幟蛋白質分子－癌胚抗原。該系統利用硫化鎘量子點作為訊號源，並利用奈米碳球增強訊號，進而增高對癌胚抗原之偵測靈敏度，適合用於拋棄式居家照護之自我檢測。該生化感測器基於三明治型免疫分析法之原則，依序在電極上修飾癌胚抗原抗體，隨後與樣品中之癌胚抗原反應，最後再加入修飾有硫化鎘量子點之癌胚抗原抗體，形成三明治型免疫複合物。本研究運用方波陽極剝除伏安法放大訊號電流應答，掃描利用硝酸溶解之硫化鎘量子點的電流訊號。該分析系統對於癌胚抗原濃度之線性範圍為0.032至10 ng/mL，偵測極限為32 pg/mL (在5 L分析樣品中相當於160 fg癌胚抗原)。本研究之分析方法也適用於準確且靈敏地偵測尿液中之癌胚抗原，可用作早期偵測尿道上皮癌之腫瘤標幟。
本論文的第二部分則是利用針式過濾器與玻璃裝置，以雙重乳化法作為樣板，研發設計出一套簡便且可程序化之注入裝置，用於合成高包覆效率之微脂體。首先，水相溶液與磷脂質之氯仿溶液經由輸液幫浦注入玻璃裝置，形成W/O/W雙重乳化物。隨後利用旋轉減壓濃縮機使氯仿蒸發而形成微脂體，最後再經由透析移除未包覆之螢光染劑。利用本研究所設計之裝置製備微脂體，其包覆效率約為26%，且可藉由改變裝置中之過濾膜孔徑而得到奈米尺寸之微脂體。該裝置不需使用音波震盪器等攪拌裝置或精密之微流道系統，有助於大量製備高包覆效率之微脂體，以作為訊號分子或藥物的載體。

關鍵字

電化學免疫分析 ； 癌胚抗原 ； 奈米碳球 ； 聚乙烯亞胺 ； 網版印刷電極 ； 硫化鎘量子點 ； 方波陽極剝除伏安法 ； 針式過濾器 ； 微脂體 ； 雙重乳化法

並列摘要

In the first part of thesis, we have presented a sensitive electrochemical immunoassay system for the detection of a protein tumor marker, carcinoembryonic antigen (CEA), that is based on a carbon nanoparticle (CNP)/poly(ethylene imine) (PEI)-modified screen-printed graphite electrode (CNP–PEI/SPGE) covered with anti-CEA antibodies. The signal amplification strategy–using CdS nanocrystals as biotracers and CNPs to enhance electron transfer–improves the sensitivity and detection limit for CEA, suggesting that this system holds promise for development into a point-of-care or disposable home-care self-diagnostic tool. This biosensor is based on a sandwich complex immunoassay, which we assembled from sequential layers of the anti-CEA antibody (CEA) on CNP–PEI/SPGE, the CEA sample, and the CdS nanocrystal quantum dots (QDs) sensitized with CEA (CEA–CdS QD). We used square wave anodic stripping voltammetry (SWASV) to amplify the signal current response obtained from the dissolved CEA–CdS QDs. The calibration curve for CEA concentration was linear in the range of 0.032–10 ng/mL; the detection limit (estimated as the mean of the blank sample plus three times the standard deviation obtained on the blank sample) was 32 pg/mL (equivalent to 160 fg in a 5 L sample). This method is suitably precise and sensitive to function as a means of determining urinary CEA, which is a better marker than serum CEA for the early detection of urothelial carcinoma.
In the second part of thesis, a simple-used and programmable injection device was developed, using syringe filter and glass device, to manufacture liposomes with high encapsulation efficiency based on double emulsion template. First of all, aqueous solutions and lipids in chloroform were injected into the glass device by infusion pumps respectively to form water-in-oil-in-water double emulsions. It was followed by the removal of chloroform by rotary evaporator for converting double emulsions to liposomes. At the end of the process, non-encapsulated fluorescent dye molecules were separated from liposomes by dialysis. The encapsulation efficiencies of liposomes are around 26%, and the expected size of liposomes could be achieved by syringe filter membranes with designated pore size. This device is workable with neither sonicator nor delicate microfluidic system, and is suitable for manufacture of liposomes as carriers of signal molecules or drugs with high encapsulation efficiency.

並列關鍵字

electrochemical immunoassay ； carcinoembryonic antigen ； carbon nanoparticle ； poly(ethylene imine) ； screen-printed graphite electrode ； CdS nanocrystal quantum dot ； square wave anodic stripping voltammetry ； syringe filter ； liposome ； double emulsion template

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