合成多巴胺鍵結之碳量子點應用於分析血漿中之deferasirox

The synthesis of luminescent dopamine-conjugated carbon dots for selective detection of deferasirox in plasma

黃柏蒼(Po-Tsang Huang)

指導教授 ： 吳秀梅

高雄醫學大學/藥學院/藥學系碩士在職專班/碩士(2019年)

摘要

本研究利用合成與多巴胺鍵結之碳量子點偵測乙型地中海型貧血病人血漿中deferasirox的含量。與多巴胺鍵結之碳量子點合成時以檸檬酸和尿素為前驅物，均勻混合成水溶液後，放入微波硝化器中加熱反應。微波硝化器反應過程區分三階段，包含自室溫加熱至200℃，於200℃下維持恆溫及冷卻，每一階段各需30分鐘。反應完成後，用濾膜過濾及透析膜透析，接著於PBS 緩衝液的環境下和N-hydroxysuccinimide 及 1-(3-Dimethylaminopropyl)-3-ethylcarbodimide)反應，生成碳量子點，再加入多巴胺與其鍵結，最後將生成的產物再次透析，並冷藏保存。
合成完畢後，運用穿透式電子顯微鏡及動態光散射之粒徑分析儀來確認其粒徑大小及界達電位，並用紅外光光譜儀檢測碳量子點與多巴胺是否結合成功。結果顯示多巴胺結合成功，且其粒徑大小小於10 奈米，界達電位為-25.47 mV。本研究利用銅離子可與多巴胺之鄰苯二酚結構結合，使碳量子點螢光猝滅，及排鐵劑deferasirox (DFX)加入後，可螯合銅離子，使之避免與多巴胺結合，進而使螢光回復的原理，藉由螢光回復的程度來計算DFX 的濃度。
本研究方法的最適化條件為利用20 µM銅離子使螢光猝滅，DFX與銅離子螯合的時間為30秒，利用 800 µL 的丙酮對血漿樣品去蛋白，並於樣品中加入30% 甲醇，於pH 11下反應。本方法偵測極限為600 ng/mL，定量分析範圍為1到10 µg/mL，迴歸係數均大於0.9975。經方法確效，相對標準差(RSD)皆小於8.7%，相對標準誤差(RE)皆小於4.2%。相對回復率介於99.42%-105.74%。確效後，利用不同金屬離子與銅離子比較，及不同螯合劑與DFX比較，顯示均有良好之專一性。本方法具有樣品前處理過程簡單，分析時間短等特點。本方法可成功應用於分析5位乙型地中海型貧血病人血漿中DFX濃度，相對標準差(RDS)小於4.71%，另將其偵測結果與運用CE-UV之方法偵測的結果比較後，相對標準誤差(RE)小於4.78%，顯示此方法具有良好之精確度及準確度。

關鍵字

碳量子點 ； 去鐵劑 ； 多巴胺 ； 螢光猝滅 ； 螢光回復

並列摘要

A novel dopamine-conjugated carbon dot (DA-CD) biosensor has been designed for the estimation of deferasirox (DFX) in biological samples. The DA-CDs were synthesized by using citric acid (CA) and urea as precursors. They were first mixed and heated in the microwave to obtain the initial product. They further reacted with N-hydroxysuccinimide and 1-(3-Dimethylaminopropyl)-3-ethylcarbodimide) in the presence of PBS buffer. Afterwards, dopamine hydrochloride was added to obtain the final product.
When the synthesis was completed, the final product was characterized by its particle size, zeta potential and functional groups using transmission electron microscope (TEM), dynamic light scattering (DLS) and Fourier-transform infrared spectroscopy (FT-IR). The particle size found was below 10 nm and the zeta potential was -25.47 mV. The conjugation of dopamine increased the negative charge of the carbon dots and enabled binding with copper ions that causes quenching of fluorescence intensity. The fluorescence intensity was quenched by Cu2+ and recovered when DFX was added. Through the calculation of fluorescence recovery, the concentration of DFX was therefore determined. Plasma samples were deproteinated using acetone and the supernatants were collected for sample analysis. The optimized condition was found at Cu2+concentration 20 µM, reaction time of deferasirox with Cu2+ 30 seconds, deproteination solvent 800 µL acetone, methanol percentage 30% and pH 11. The limit of detection was 600 ng/mL. The linearity response for DFX was within the concentration range from 1µg/mL to 10 µg/mL with a correlation coefficient greater than 0.9975. All the other parameters like accuracy, precision was under ± 10 % which was under acceptable range. After the validation, the specificity of Cu2+ and deferasirox were both studied. This method offered very simple sample pre-treatment, short time analysis and was very specific. The method was further applied to biological samples and the data was compared with those found by CE-UV. The RSD were below 4.71, and the RE were below 4.78, which were both in acceptable range.

並列關鍵字

carbon dots ； deferasirox ； dopamine ； fluorescence quenching ； fluorescence recovery

參考文獻

1. Anthony T; George W, Isao K Biosensors: Fundamentals and Applications. Oxford, UK: Oxford University (1987). Press. Page. 770.

2. Bănică, Florinel-Gabriel. Chemical Sensors and Biosensors: Fundamentals and Applications. Chichester, UK: John Wiley & Sons. (2012) Page. 576.

3. Eitan F, Eliezer A. R. Iron overload in hematological disorders. La Presse Médicale. (2017) 46, e296-e305.

4. Griffiths, W. J. H. Haemochromatosis. Medicine, (2011) 39, 597–601.

5. Ali T. T and Antoine N. S. Iron overload in thalassemia: different organs at different rates. Hematology. 2017 (1), 265- 271