利用不勻相的形成目視偵測特定濃度範圍鐵離子

Abstract

本論文以目視觀察相變化(phase segregation)偵測特定濃度範圍的鐵離子。反應試劑包含imidazole緩衝溶液及具有硫醇官能基(－SH)之青黴胺(penicillamine)，與鐵離子產生棉絮狀物質，使溶液呈不勻相，利用鐵離子與青黴胺之莫耳數比值((mFe3+/mpenicillamine)0)可界定出三個相(phases)的變化：當(mFe3+/mpenicillamine)0小於0.3時，形成不勻相需時超過20分鐘(phase I)；當(mFe3+/mpenicillamine)0介於0.3~1.0時，20分鐘內形成不勻相(phase II)；當(mFe3+/mpenicillamine)0大於1.0時，溶液不形成不勻相(phase III)。此分析方法的目標樣品為人體血清(人體血清鐵離子的正常濃度範圍：9.0~31.3 uM)，探討其他金屬離子是否對本分析鐵離子系統造成影響，實驗發現只有[Cu2+]0會影響反應試劑與鐵離子形成不勻相之濃度範圍，但在Cu2+存在時，仍可用(mFe3+/mpenicillamine)0區別三相變化，且可更快速觀察到不勻相的形成，如phase II於2分鐘內形成不勻相。樣品濃度達mM時，可直接以肉眼觀察到不勻相的形成；而當樣品濃度為uM時，不勻相的形成量減少，肉眼辨識困難，應用庭得耳效應(Tyndall effect)可利用雷射投影筆產生之光徑幫助肉眼觀察相變化。藉由觀察2分鐘內不勻相形成與否，判斷鐵離子的濃度範圍。實驗參數如pH值、溫度及離子強度亦會影響不勻相的形成，推測原因與imidazole的pKa、反應活化能及不勻相之表面電荷斥力有關。在參數最佳化的條件下，此方法可簡單、快速判定人體血清鐵離子濃度是否正常。為了瞭解形成不勻相之原因，利用國家同步輻射中心的X光吸收光譜、還原劑ascorbic acid及螯合劑ferrozine (針對Fe2+)與bathocuproinedisulfonic acid disodium salt (bcs，針對Cu+)探討形成不勻相之反應機制。

Visual detection triggered by sensing reagents has been one of the central themes in contemporary chemistry. This thesis work develops a rapid screening scheme to determine whether the amount of Fe3+ falls into a diagnostic concentration range (9.0~31.3 uM) in human serum samples by the naked eye. It is important for applications under circumstances where instruments are not readily available. The goal is achieved by the formation of flocculates within a certain range of Fe3+-to-penicillamine mole ratios ((mFe3+/mpenicillamine)0). Experimental results show that flocculates formed less than 2 minutes for (mFe3+/mpenicillamine)0 ranging from 0.3 to 1.0 when Cu2+ exists. Outside the range, the solution appears homogeneous. With a handy laser pointer, Tyndall effect that the light is scattered by colloidal suspension is adapted to realize semi-quantification of Fe3+. The reaction depends on solution pH, temperature, ionic strength, and (mFe3+/mpenicillamine)0 mole ratios. Under the optimized experimental conditions, the visual sensing method is applied to serum Fe3+ detection. To investigate the mechanisms of forming flocculates, X-ray absorption spectrometer, reducing agent (ascorbic acid for Fe3+) and chelators (ferrozine for Fe2+, bathocuproinedisulfonic acid disodium salt for Cu+) were used in this study.