A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)
Abstract
:1. Introduction
2. Experimental
2.1. Enzymes and Reagents
2.2. Apparatus and Equipment
2.3. Principle and Measurement Protocol
2.4. Experimental Data Processing
- I% = 100 (hυ1 − hυ2) / hυ1
- hυ1 = Photon count in absence of Hg(II) compound.
- hυ2 = Photon count in presence of Hg(II) compound.
2.5. Sample Preparation
3. Results and Discussion
3.1. Enzymatic Assay Development: for Bioassay Development the Following Parameters Were Optimized
3.1.1. Effect of Ionic Strength
3.1.2. Effect of pH
3.1.3. Effect of Enzyme Concentration
3.1.4. Effect of Temperature
3.1.5. Optimization of Substrate Specificity and Substrate Concentration
3.2. Inhibition Studies for Heavy Metal Determination
3.2.1. Hg(II) Determination
Effect of Incubation Time
Effect of Inhibitor Concentration
3.2.2. Lead and Cadmium Determination
Inhibition by Cd(II) and Pb(II)
Mixture of Hg(II), Cd(II) and Pb(II)
3.3. Analysis in Real Samples
3.4. Miniaturization of Enzymatic Assay and Highly Sensitive Determination of Hg(II)
3.4.1. Effect of Miniaturization on Substrate Response in 96 and 384 Well Plate Format
3.4.2. Effect of Miniaturization on Inhibition Studies in 96 and 384 Well Plate Format
3.5. Stability of the Enzyme and Reproducibility of the Assay in Well Plate
4. Conclusions
Acknowledgments
References
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Parameter | Experimental range |
---|---|
Enzyme concentration | 0.00075–0.024 U/assay |
Substrate specificity | Methanol, ethanol and propanol 10 nM to 1 M |
pH of buffer | 6.8–9.0 |
Ionic conductivity of buffer | 0.01–0.2 M |
Temperature | 28–40 °C |
Analyte | Hg (II) |
---|---|
Linear dynamic range | 0.005–50 ng·mL−1 |
Analysis time | 25 min |
Apparent IC50 | 0.3 ng·mL−1 |
R2 | 0.9949 |
Minimum Detection limit | 2.5 pg·mL−1 |
Total assay volume | <100 μL |
Throughput | about 70 samples in less than 30 min (leaving blank and reference) |
Matrix | Spiked Hg(II) (pg·mL−1) | Hg(II) found (pg·mL−1) | Difference (pg·mL−1) | % Recovery |
---|---|---|---|---|
Drinking Water | 75 | 75 | ----- | 100 |
Tap water | 75 | 77 ± 0.05 | 2.05 | 102.66 |
Sea water-1 | 75 | 80 ± 0.75 | 5.75 | 106.66 |
Sea water-2 | 75 | 79 | 4 | 105.33 |
Sea water-3 | 75 | 82± 0.89 | 7.89 | 110.52 |
Parameter | 96 well-plate assay | 384 well plate assay |
---|---|---|
Enzyme U/assay | 0.01 | 0.002 |
Incubation time, min | 20 | 10 |
IC30 (pg·mL−1) | 60 | 32 |
Total assay volume (μL) | 100 | 20 |
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Deshpande, K.; Mishra, R.K.; Bhand, S. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II). Sensors 2010, 10, 6377-6394. https://doi.org/10.3390/s100706377
Deshpande K, Mishra RK, Bhand S. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II). Sensors. 2010; 10(7):6377-6394. https://doi.org/10.3390/s100706377
Chicago/Turabian StyleDeshpande, Kanchanmala, Rupesh K. Mishra, and Sunil Bhand. 2010. "A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II)" Sensors 10, no. 7: 6377-6394. https://doi.org/10.3390/s100706377
APA StyleDeshpande, K., Mishra, R. K., & Bhand, S. (2010). A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II). Sensors, 10(7), 6377-6394. https://doi.org/10.3390/s100706377