痕量葡萄糖的双酶催化荧光分析

在HAe-NaAc缓冲溶液中,葡笱糖氧化酶(GOD)催化葡萄糖与溶解氧反应牛成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.在最佧条件下,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△F=40.0c+3.0,△F=23.9c+8.1,△F=25.6c+4.2,△F=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.

Fluorescence Analysis of Trace Glucose Using Glucose Oxidase and Horseradish Peroxidase

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.