| 光谱学与光谱分析 |
|
|
|
|
|
| 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%.
|
|
Received: 2007-11-16
Accepted: 2008-02-26
|
|
|
|
Corresponding Authors:
JIANG Zhi-liang
E-mail: zljiang@mailbox.gxnu.edu.cn
|
|
[1] Sun W, Jiao K, Zhang S S, et al. Anal. Chim. Acta, 2001, 434: 43.
[2] Fanjul-Bolado P, Gonzalez-Garcia M B, Costa-Garcia A. Talanta, 2004, 64(2): 452.
[3] Jiao K, Sun G, Zjhang S S. Sci. Chin. Series B，1998，28：157.
[4] YANG Wei-ping, ZHANG Zhu-jun, LI Bao-lin(杨维平，章竹君，李宝林). Journal of Shaanxi Normal University(陕西师范大学学报), 1995，23(2)：59.
[5] Riko I, Hiroshi I, Takashi H, et al. Anal. Biochem., 1995, 231: 170.
[6] Hashem A T, Katsuaki S, Zahra A, et al. Clinical Chem., 1995, 41: 1368.
[7] Luciana M K, Anderson A M, Alexandre K, et al. Bioorg & Med. Chem., 2007, 15: 1516.
[8] WEI Yong-feng, YAN Hong-tao(魏永锋，阎宏涛). Spectroscopy and Spectral Analysis(光谱学与光谱分析)，2001，21(5)：704.
[9] WANG Tong, LI Zeng(王 彤, 李 增). Chemical Reagents(化学试剂), 2005, 27(3): 155.
[10] Rad A M, Ghourchian H, Moosavi-Movahedi A A, et al. Anal. Biochem., 2007, 362: 38.
[11] Bovaird J H, Ngo T T, Lenhoff H M. Clinical Chem., 1982，28: 2423.
[12] WEI Yong-feng, YAN Hong-tao(魏永锋，阎宏涛). Chin. J. Anal. Chem.(分析化学)，2000，28：99.
[13] ZHANG Shu-sheng, JIAO Kui, JI Xian-feng(张书圣，焦 奎，季先锋). Journal of Qingdao Chemical Engineering Institute(青岛化工学院学报)，1997，18(4)：333.
[14] Peter W A, Carmelita M. Anal. Chim. Acta, 1988, 208: 173.
[15] Eloy S, Angel A J, Torriero M I, et al. Talanta, 2005, 66: 92.
[16] LIANG Ai-hui, JIANG Zhi-liang, ZHOU Su-mei, et al(梁爱惠，蒋治良，周苏梅，等). Spectroscopy and Spectral Analysis(光谱学与光谱分析)，2006，26(11): 2113.
[17] JIANG Zhi-liang, LI Fang, LIANG Hong(蒋治良，李 芳，梁 宏). Acta Chim. Sin.(化学学报)，2000，58: 1059.
[18] Molaei A Rad, Ghourchian H, Moosavi-Movahedi A A, et al. Anal. Biochem., 2007, 362: 38.
[19] LIU Bao-sheng，GAO Jing，LIU Zhi-chao，et al(刘保生，高 静，刘智超，等). Spectroscopy and Spectral Analysis(光谱学与光谱分析)，2006，26(2)：306.
|
| [1] |
TANG Qian1, 3*, GONG Ting-ting1,3, CAO Hong-yu1, 3, WANG Li-hao2,3, LI Ru-yu2, ZHENG Xue-fang1, 2, 3*. Mechanism of the Variation of Horseradish Peroxidase Catalytic Activity Induced by Light[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2018, 38(12): 3692-3698. |
| [2] |
QIU Xin1, LI Xin-xia2, Arkin IBURAIM2, Gulina KUERBAN3, ZHANG Li-hua1, LI Yan1*. Determination of Cephradine Capsules Content Based on Fluorescence Quenching Method of FOS-μSIA-LOV[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2017, 37(11): 3475-3478. |
| [3] |
YANG Cheng-hu1, LIU Yang-zhi1, ZHU Ya-xian2, ZHANG Yong1，3*. In Situ Investigation of the Interactions of Pyrene and Phenanthrene with Humic Acid Using Laser Induced Nanoseconds Time Resolved Fluorescence Quenching Method Combined with PARAFAC Analysis[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2017, 37(05): 1519-1524. |
| [4] |
LIU Yang-zhi1, YANG Cheng-hu1, ZHU Ya-xian2, ZHANG Yong1，3* . In Situ Investigating of the Interaction of Phenanthrene and Alkyl Phenanthrene with Humic Acid with Laser Induced Nanoseconds Time Resolved Fluorescence Quenching Method[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2016, 36(10): 3332-3336. |
| [5] |
ZHANG Qiu-ju, LIU Bao-sheng*, LI Gai-xia, HAN Rong, Lü Yun-kai . The Investigation on the Interaction of Colistin Sulfate with Bovine Serum Albumin with Resonance Light Scattering Spectroscopy [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2016, 36(09): 2879-2883. |
| [6] |
ZHONG Wen-ying1, 2, WANG Yan1, HUANG Bin1, SHU Chang1. The Quantification of Gemifloxacin by Fluorescence Quenching Method Using Chitosan-Coated CdTe Quantum Dots [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2012, 32(06): 1570-1574. |
| [7] |
WEI Xiao-ling, LEI Xiang-rong, GONG Qi, WANG Li-sheng, LIAO Yuan . Determination of Cetirizine Dihydrochloride by Anti-Fluorescence Quenching on Rhodamine B-Sodium Tetraphenylborate System [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2011, 31(06): 1596-1600. |
| [8] |
LI Hai-tao1,2, LI Yu-ping2, CAO Hong-bin2*, LI Xin-gang1, XUE Zhan-qiang3 . The Production of Hydroxyl Radical in HRP-NADH-H2O2 /O2 Systems and Its Application in Chlorobenzene Removal [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2010, 30(11): 3119-3123. |
| [9] |
MA Jin-jie, LIANG Jian-gong, HAN He-you* . Study on the Synchronous Interactions between Different Thiol-Capped CdTe Quantum Dots and BSA [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2010, 30(04): 1039-1043. |
| [10] |
LI Ye, YANG Hui, HAN Wei-wei, LIAO Ming-xia, LU Yi-qiang. An Enzyme Sensor for Phenolic Compounds Analysis[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2010, 30(02): 571-574. |
| [11] |
LIU Qing-ye1, LIANG Yue-yuan1, LIANG Ai-hui2, JIANG Zhi-liang1, 2* . Fluorescence Analysis of Trace Glucose Using Glucose Oxidase and Horseradish Peroxidase[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(09): 2535-2538. |
| [12] |
LIU Yi-ming,XUAN Chun-sheng,LI Wen-ying*,FENG Jie. Study of Cefadroxil and Cephradine Charge Transfer Process by Fluorescence Quenching Method[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2009, 29(02): 441-445. |
| [13] |
LI Zhong-qin1,2,XU Xiao-ping1,2*,WANG Wu1. Catalytic Spectrophotometric Quantitation for Hypoxanthine by Conjugating Xanthine Oxidase with Horseradish Peroxidase[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2008, 28(09): 2169-2172. |
| [14] |
XU Qiang1,YANG Ti-qiang1*,AO Dun-ge-ri-le1,YAO Zhan-quan2 . Study on the Effect of Electric Field on the Secondary Structure of HRP by Circular Dichroism [J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2007, 27(03): 565-568. |
| [15] |
LIANG Ai-hui1, JIANG Zhi-liang1*, ZHOU Su-mei2, ZHOU Yu-chan2, LIANG Yue-yuan2, CHEN Li-yu2 . Rhodamine 6G Association Particle Based Spectrophotometric Determination of Hydroxy Free Radical and Its Application to the Selection of Antioxidant[J]. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2006, 26(11): 2113-2115. |
|
|
|
|
