The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol
Abstract
:1. Introduction
2. Materials and Methods
2.1. Apparatus
2.2. Reagents
2.3. Procedures
2.3.1. Single-Solution Protocol
2.3.2. Modified Single-Solution Protocol
2.3.3. Two-Stage Protocol
3. Results and Discussion
3.1. The Stoichiometry in the Reaction between Hydrogen Peroxide and Scopoletin
3.2. The Effect of pH on the Reaction between Hydrogen Peroxide and Scopoletin
3.3. Photobleaching of Scopoletin
3.4. Deactivation of Horseradish Peroxidase
3.5. Sample Storage
3.6. Comparison of Results Obtained from the Single-Solution Protocols and the Two-Stage Protocol
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Fluorometer | C0 (µM ) | k (10−4 s−1) | r |
---|---|---|---|
Hitachi Model F7000 | 2.003 | 3 | 0.9987 |
1.487 | 3 | 0.9895 | |
0.994 | 3 | 0.9960 | |
0.493 | 3 | 0.9835 | |
Turner Model10-AU | 0.980 | 0.13 | 0.9375 |
0.494 | 0.13 | 0.9465 |
Sample | Specific Fluorescence (f.u. μM−1) | |
---|---|---|
Without Additional HRP | With Additional HRP | |
RW | 357, 369 | 370, 379 |
RW + 0.1 μM H2O2 | 334, 333 | 367, 361 |
Average | 348 ± 18 | 369 ± 8 |
SW | 438, 438 | 582, 609 |
SW + 0.1 μM H2O2 | 306, 305 | 568, 590 |
Average | 372 ± 77 | 587 ± 17 |
Sample | H2O2 Added μM | Protocol A Found μM | Protocol B Found μM | B/A % | Protocol C Found μM | C/A % |
---|---|---|---|---|---|---|
RW | 0 | 0.013, 0.011 | ||||
Average | 0.012 ± 0.001 | |||||
0.10 | 0.116, 0.115 | 0.120, 0.121 | 0.158, 0.152 | |||
Average | 0.116 ± 0.001 | 0.120 ± 0.001 | 103 | 0.155 ± 0.005 | 134 | |
Recovery | 0.104 ± 0.002 | |||||
% Recovery | 104 ± 2 | |||||
RW | 0 | 0.021, 0.020 | ||||
Average | 0.021 ± 0.001 | |||||
0.10 | 0.120, 0.118 | 0.128, 0.122 | 0.156, 0.162 | |||
Average | 0.119 ± 0.001 | 0.125 ± 0.004 | 105 | 0.159 ± 0.005 | 134 | |
Recovery | 0.098 ± 0.002 | |||||
% Recovery | 98 ± 2 | |||||
SW | 0 | 0.024, 0.021 | ||||
Average | 0.023 ± 0.002 | |||||
0.10 | 0.130, 0.124 | 0.158, 0.161 | 0.291, 0.271 | |||
Average | 0.127 ± 0.003 | 0.159 ± 0.002 | 125 | 0.281 ± 0.014 | 221 | |
Recovery | 0.104 ± 0.005 | |||||
% Recovery | 104 ± 5 | |||||
SW | 0 | 0.022, 0.016 | ||||
Average | 0.019 ± 0.004 | |||||
0.10 | 0.121, 0.127 | 0.146, 0.154 | 0.285, 0.290 | |||
Average | 0.124 ± 0.004 | 0.150 ± 0.005 | 121 | 0.288 ± 0.004 | 232 | |
Recovery | 0.105 ± 0.008 | |||||
% Recovery | 105 ± 8 | |||||
SW | 0 | 0.015, 0.012 | ||||
Average | 0.014 ± 0.002 | |||||
0.10 | 0.114, 0.117 | 0.140, 0.150 | 0.219, 0.252 | |||
Average | 0.111 ± 0.005 | 0.145 ± 0.007 | 130 | 0.235 ± 0.023 | 212 | |
Recovery | 0.097 ± 0.007 | |||||
% Recovery | 97 ± 7 |
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Wu, M.; Wong, G.T.F.; Wu, Y.-C. The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol. Methods Protoc. 2018, 1, 4. https://doi.org/10.3390/mps1010004
Wu M, Wong GTF, Wu Y-C. The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol. Methods and Protocols. 2018; 1(1):4. https://doi.org/10.3390/mps1010004
Chicago/Turabian StyleWu, Man, George T. F. Wong, and Yao-Chu Wu. 2018. "The Scopoletin-HRP Fluorimetric Determination of H2O2 in Seawaters—A Plea for the Two-Stage Protocol" Methods and Protocols 1, no. 1: 4. https://doi.org/10.3390/mps1010004