罗丹明染料荧光猝灭法测定超痕量辣根过氧化物酶

Fluorescence Quenching Assay of Ultratrace Horseradish Peroxidase Using Rhodamine Dye

在HAC-NaAC缓冲液中，辣根过氧化物酶催化H2O2氧化KI生成I2，过量的I^-可与I2结合形成I3，而I3^-分别与罗丹明S（RhS），罗丹明G（Rh6G），罗丹明B（RhB）和丁基罗丹明B（b-RhB）反应导致四体系在580，580，554和554nm处的荧光强度降低。在选择条件下，对于RhS，Rh6G，RhB，b-RhB四体系，辣根过氧化物酶的浓度分别在8～6400，40～4000，32～3200，40～6400Pg·mL^-1范围内与其荧光猝灭强度成线性关系，其检出限分别为3．2，3．0，2．4，3．7pg·mL^-1。其中RhS催化体系较好，用于酶联免疫乙肝试剂盒中辣根过氧化物酶活力的测定，结果比较满意。

In acetate buffer solution, the reaction of H2O2 with KI was catalyzed by horseradish peroxidase （HRP） to form I3^- The I3^- 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 （△F） 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^-1KI-1.30×10^-5s mol ·L^-1 H2O2-25℃-20 min, 4.8-2.4×10^-5 mol·L^-1 Rh6Cr4× 10^-3 mol ·L^-1 KI-2.59×10^-5 mol·L^-1 H202-25 ℃- 20min, 4.6-1.6×10^-5s mol·L^-1 RhB4×10^-3 mol· L^-1 KI-2.16×10^-5 mol· L ^-1 H2O2-25℃-20min, 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 △F=0. 061 1c＋39.6, △F=0. 047 2c＋50. 4, △F=0. 138 6c＋34. 2 and△F=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, Ca^2＋ , Mg^2＋ , Cu^2＋ , Fe^3＋ , Zn^2＋ and vitamin 136, 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%.