Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Instrumentation
2.3. Probe 1 Selection
2.4. Label-Free K-Ras Activity Assays
2.5. QRET-Based Control Assay for GTPase Cycling and GTP Association Monitoring
2.6. Label-Free Apyrase ATPase Activity Monitoring
2.7. Data Analysis
3. Results and Discussion
3.1. Label-Free Terbium(III)-Probe Enables Nanomolar Detection of Phosphate-Containing Molecules
3.2. K-Ras Functionality Can Be Monitored at Nanomolar Sensitivity Similar to the Control Assay
3.3. Probe 1 Enables Nucleotide Triphosphate Hydrolysis Monitoring to Di- and Further to Nucleotide Monophosphates
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Method | Detection | Reaction | Assay Buffer | Measurement |
---|---|---|---|---|
GTPase cycling assay | Probe 1: 7.5 µM antenna 1, 7.5 nM Tb(III)-N1 QRET: 7.5 nM Eu(III)-GTP, 12 nM 2A4GTP Fab, 1.8 µM MT2 | 200 nM K-Ras, 200 nM SOScat, 100 nM GAP (p120RasGAP or NF1), 1.5 µM GTP | Buffer 1: 20 mM HEPES, pH 7.5, 1 mM MgCl2, 0.01% Triton-X 100, 0.005% γ-globulins | Probe 1: 330/545 nm Tb(III)-luminescence (10 µL) QRET: 340/615 nm Eu(III)-luminescence (10 µL) |
GTP association assay | Probe 1: 7.5 µM antenna 1, 7.5 nM Tb(III)-N1 QRET: 10 nM Eu(III)-GTP, 1.5 µM MT2 | 500 nM K-Ras, 250 nM SOScat, 500 nM GTP 200 nM K-Ras, 200 nM SOScat, | Buffer 1: 20 mM HEPES, pH 7.5, 1 mM MgCl2, 0.01% Triton-X 100, 0.005% γ-globulins | Probe 1: 330/545 nm Tb(III)-luminescence (10 µL) QRET: 340/615 nm Eu(III)-luminescence (10 µL) |
Apyrase assay | Probe 1: 7.5 µM antenna 1, 7.5 nM Tb(III)-N1 | 50 µU apyrase, 1–10 µM ATP | Buffer 2: 20 mM HEPES, pH 6.5, 1 mM MgCl2, 1 mM CaCl2, 0.01% Triton-X 100, 0.005% γ-globulins | Probe 1: 330/545 nm Tb(III)-luminescence (10 µL) |
Anion Species | EC50 (nM) | LOD (nM) | Linear Range (µM) |
---|---|---|---|
ATP | 107 ± 0.5 | 15.7 ± 3.0 | 0.02–5 |
GTP | 97 ± 0.4 | 18.2 ± 2.5 | 0.02–10 |
CTP | 17 ± 0.2 | 3.1 ± 1.5 | 0.01–1 |
ITP | 155 ± 0.4 | 43.6 ± 5.7 | 0.02–10 |
UTP | 164 ± 0.7 | 28.2 ± 4.4 | n.c. |
AMP-PNP | 73 ± 0.6 | 8.6 ± 3.9 | n.c. |
ADP | 1170 ± 70 | 161 ± 48 | 0.2–150 |
GDP | 1110 ± 60 | 197 ± 42 | 0.2–100 |
UDP | 1540 ± 70 | 165 ± 36 | 0.2–150 |
GMP | 16,800 ± 3900 | n.c. | n.c. |
(NaPO3)n | 33 ± 0.2 | 13.6 ± 7.2 | n.c. |
(NaPO3)3 | 60 ± 0.4 | 15.5 ± 2.6 | n.c. |
(NaPO3)2 | 432 ± 3 | 69.3 ± 20.2 | n.c. |
Na2HPO4 | >250 | n.c. | n.c. |
cGMP | n.d. | n.d. | n.d. |
cAMP | n.d. | n.d. | n.d. |
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Kopra, K.; Seppälä, T.; Rabara, D.; Abreu-Blanco, M.; Kulmala, S.; Holderfield, M.; Härmä, H. Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content. Sensors 2018, 18, 3989. https://doi.org/10.3390/s18113989
Kopra K, Seppälä T, Rabara D, Abreu-Blanco M, Kulmala S, Holderfield M, Härmä H. Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content. Sensors. 2018; 18(11):3989. https://doi.org/10.3390/s18113989
Chicago/Turabian StyleKopra, Kari, Tanja Seppälä, Dana Rabara, Maria Abreu-Blanco, Sakari Kulmala, Matthew Holderfield, and Harri Härmä. 2018. "Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content" Sensors 18, no. 11: 3989. https://doi.org/10.3390/s18113989
APA StyleKopra, K., Seppälä, T., Rabara, D., Abreu-Blanco, M., Kulmala, S., Holderfield, M., & Härmä, H. (2018). Label-Free Time-Gated Luminescent Detection Method for the Nucleotides with Varying Phosphate Content. Sensors, 18(11), 3989. https://doi.org/10.3390/s18113989