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

在HAc-NaAc缓冲溶液中，葡萄糖氧化酶（COD）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584nm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0．0839．99，0．17～8．33，0．33～8．33，0．33～9．99μmol·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；0．9951，0．9973，0．9960，0．9965；0．059，0．17，0．21，0．16μmol·L^-1RhS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。

In acetate buffer solution and in the presence of glucose oxidase （GOD）, glucose reduced the dissolved oxygen to form H2 02 that oxidized catalytically the excess KI to from I3- by horseradish peroxidase （HRP）. The I3- combines respectively with rhodamine S （RhS）, rhodamine 6G（Rh6G）, butyl-rhodamine B（b-RhB） and rhodamine B（RhB） to form RhS-Ia, 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. 33-9.99μmol·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. 16μmol·L^-1 glucose. And the regression equation was △F=40.0c＋3.0,△F=23.9c＋8.1,△F=25.6c＋4.2,△F=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 ~ 10M. Results showed that 1 000-fold Mg2＋ and Cu2＋ , 300-fold Mn2＋ , 100-fold Zn2＋ , AP＋ and Co2＋ , 60-fold L-tyrosine, urea and nicotinic acid, 50-fotd Fe3＋ , HSA and BSA, 10-fold sucrose, vitamin 132, L-lysine, L-glutamic acid and L-cystine did not interfere with the determinatiorL This RhS fluorescence quenching assay was applied to the determination of glucose in the serum samples with satisfactory results.