Fluorescence Analysis of Trace Glucose Using Glucose Oxidase and Horseradish Peroxidase
LIU Qing-ye1, LIANG Yue-yuan1, LIANG Ai-hui2, JIANG Zhi-liang1, 2*
1. School of Environment and Resource, Guangxi Normal University, Guilin 541004, China 2. Department of Material and Chemical Engineering, Guilin University of Technology, Guilin 541004, China
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.
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