Fluorescence Quenching Assay of Ultratrace Horseradish Peroxidase Using Rhodamine Dye
MA Wen-sheng1,3,HUANG Guo-xia1,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 3. Guangxi Polytechnic Institute, Nanning 530226, China
Abstract：In acetate buffer solution, the reaction of H2O2 with KI was catalyzed by horseradish peroxidase (HRP) to form I-3. The I-3 combined respectively with rhodamine S(RhS), rhodamine 6G(Rh6G), rhodamine B(RhB) and butyl-rhodamine B(b-RhB) to form RhS-I3,Rh6G-I3,RhB-I3 and b-RhB-I3 association particles, resulting in the fluorescence quenching at 580, 580, 554 and 554 nm, respectively. The effect of pH value, rhodamine dye concentration, KI concentration, H2O2 concentration, reaction temperature and time on the fluorescence quenching intensity (ΔF) of the four catalytic systems was considered respectively. For the RhS, Rh6G, RhB and b-RhB catalytic systems, pH 4.6-3.2×10-5 mol·L-1 RhS-4×10-3 mol·L-1 KI-1.30×10-5 mol·L-1 H2O2-25 ℃-20 min, 4.8-2.4×10＿5 mol·L-1 Rh6G-4×10-3 mol·L-1 KI-2.59×10-5 mol·L-1 H2O2-25 ℃-20 min, 4.6-1.6×10-5 mol·L-1 RhB-4×10-3 mol·L-1 KI-2.16×10-5 mol·L-1 H2O2-25 ℃-20 min, and 4.6-1.6×10-5 mol·L-1 b-RhB-4×10-3mol·L-1 KI-3.02×10-5mol·L-1 H2O2-25 ℃-20 min were chosen for use respectively. Under the optimal conditions, the HRP linear range was 8-6 400 pg·mL-1 for the RhS catalytic system, 40-4 000 pg·mL-1 for the Rh6G catalytic system, 32-3 200 pg·mL-1 for the RhB catalytic system and 40-6 400 pg·mL-1 for the b-RhB catalytic system, with a detection limit of 3.2, 3.0, 2.4 and 3.7 pg·mL-1 HRP, respectively. The regress equation of the four catalytic systems was ΔF=0.061 1c+39.6, ΔF＝0.047 2c+50.4, ΔF＝0.138 6c+34.2 and ΔF＝0.026 25c+36.72, with a correlation coefficient of 0.997 9, 0.999 0, 0.997 3 and 0.996 9, respectively. The RhS catalytic system was most sensitive, and was chosen for the determination of HRP. The influence of foreign substance on the RhS assay of 3.5 ng·mL-1 HRP was examined, with a relative error of ±10%. A 3000-times L-glutamic acid, L-lysine, Ca2+,Mg2+,Cu2+,Fe3+,Zn2+ and vitamin B6,1000-times HAS etc did not interfere with the assay. This showed that the assay has good selectivity. The RhS fluorescence quenching assay was applied to the determination of HRP in the solution of hepatitis B surface antibody labeling HRP, with satisfactory results. The HRP content was (13.6±0.5)ng·mL-1 HRP. The recovery was in the range of 99%-108%.
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