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痕量葡萄糖的双酶催化荧光分析
Fluorescence Analysis of Trace Glucose Using Glucose Oxidase and Horseradish Peroxidase
【摘要】 在HAc-NaAc缓冲溶液中,葡萄糖氧化酶(GOD)催化葡萄糖与溶解氧反应生成H2O2;辣根过氧化物酶(HRP)催化H2O2氧化过量的KI生成I3-,I3-分别与罗丹明S(RhS),罗丹明6G(Rh6G),丁基罗丹明B(b-RhB),罗丹明B(RhB)结合形成缔合物微粒,使得4体系分别在556,556,584和584 nm处的荧光峰强度线性降低。在最佳条件下,葡萄糖的浓度分别在0.083~9.99,0.17~8.33,0.33~8.33,0.33~9.99μmol.L-1范围内与RhS,Rh6G,b-RhB,RhB四体系的荧光猝灭强度呈良好的线性关系,其回归方程、相关系数、检出限分别为ΔF=40.0c+3.0,ΔF=23.9c+8.1,ΔF=25.6c+4.2,ΔF=18.4c+0.8;0.995 1,0.997 3,0.996 0,0.996 5;0.059,0.17,0.21,0.16μmol.L-1。RhS催化体系最灵敏、稳定,将其用于人血清中葡萄糖的检测,结果满意。
【Abstract】 In acetate buffer solution and in the presence of glucose oxidase(GOD),glucose reduced the dissolved oxygen to form H2O2 that oxidized catalytically the excess KI to from I-3 by horseradish peroxidase(HRP).The I-3 combines respectively with rhodamine S(RhS),rhodamine 6G(Rh6G),butyl-rhodamine B(b-RhB) and rhodamine B(RhB) to form RhS-I3,Rh6G-I3,b-RhB-I3 and RhB-I3 associated particles that result in fluorescence quenching at 556,556,584 and 584 nm,respectively.Under the optimal conditions,the concentration of glucose in the range of 0.083-9.99,0.17-8.33, 0.33-8.33 and 0.33-9.99 μmol·L-1 is linear with their fluorescence quenching at 556,556,584 and 584 nm,with detection limits of 0.059,0.17,0.21 and 0.16 μmol·L-1 glucose.And the regression equation was ΔF=40.0c+3.0,ΔF=23.9c+8.1,ΔF=25.6c+4.2,and ΔF=18.4c+0.8,respectively.The RhS system was the most sensitive and stable,and was chosen for use.Influence of some foreign substances on the RhS fluorescence quenching determination of 6.67 μmol·L-1 glucose was examined,with a relative error of ±10%.Results showed that 1 000-fold Mg2+ and Cu2+,300-fold Mn2+,100-fold Zn2+,Al3+ and Co2+,60-fold L-tyrosine,urea and nicotinic acid,50-fold Fe3+,HSA and BSA,10-fold sucrose,vitamin B2,L-lysine,L-glutamic acid and L-cystine did not interfere with the determination.This RhS fluorescence quenching assay was applied to the determination of glucose in the serum samples with satisfactory results.
【Key words】 Glucose; Rhodamine dye; Enzymatic catalysis; Fluorescence quenching;
- 【文献出处】 光谱学与光谱分析 ,Spectroscopy and Spectral Analysis , 编辑部邮箱 ,2009年09期
- 【分类号】Q53
- 【被引频次】3
- 【下载频次】151