Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents
2.2. Expression and Purification of Luciferase Enzymes
2.3. Preparation of Gold Nanoparticles (Au NPs)
2.4. Preparation of Silver Nanoparticles (Ag NPs)
2.5. Preparation of Core–Shell Au–Ag Nanoparticles
2.6. Preparation of BSA–Au Nanoclusters (BSA-AuNCs)
2.7. Preparation of Gold Nanorods (GNRs)
2.8. Immobilization of Enzyme with Metal-NPs
2.9. Luciferase Activity Assay
2.10. UV/Visible Spectra
2.11. Particle Size Distribution and Zeta Potential
2.12. Transmission Electron Microscopy (TEM)
2.13. Fluorescence Spectra Measurement
2.14. Circular Dichroism (CD) Analysis
2.15. Bioluminescence Spectra (BL Spectra)
2.16. AuNP Stability Test
2.17. Adenosine Triphosphate (ATP) Assay
2.18. Cell Culture
3. Results
3.1. Nanoparticle Characterization
3.2. Nanoparticle Effect on Luciferase Activity
3.3. The Enzyme–Substrate Complexes Effect on SPR
3.4. Nanoparticle Effect on Structural Properties
3.5. AuNP Stability Test
3.6. ATP Assay
3.7. Cell Culture
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Samples | α-Helix (%) | β-Strand (%) |
---|---|---|
Fluc | 84.27 | 1.24 |
Fluc-AgNPs | 67.45 | 3.24 |
Rluc | 47.93 | 8.19 |
Rluc-Au-AgNPs | 43.94 | 10.56 |
Mfluc | 19.69 | 31.52 |
Mfluc-GNRs | 12.96 | 38.78 |
Size | Zeta | |||||||
---|---|---|---|---|---|---|---|---|
NPs | Rluc | Fluc | Mfluc | NPs | Rluc | Fluc | Mfluc | |
Au NPs | 29.0 | 183.0 | 290.0 | 72.0 | −28.0 | +13.0 | +6.0 | −7.0 |
Ag NPs | 13.5 | 82.0 | 44.0 | 153.3 | −24.0 | −9.0 | −6.0 | −8.0 |
Au-Ag NPs | 3.0 | 55.0 | 412.0 | 80.12 | −33.0 | −9.0 | −6.0 | −9.0 |
BSA-Au NCs | 31.0 | 119.0 | 98.0 | 52.04 | −39.0 | −12.0 | −4.0 | −6.0 |
GNRs | 49.0 | 589.0 | 400 | 1529.0 | +15.0 | −1.0 | +2.0 | −5.0 |
Enzymes | - | 68.0 | 61.0 | 69.0 | - | −2.0 | −9.0 | −4.0 |
Methods/Materials Used | Analytical Ranges/LODs (μM) | Samples | References |
---|---|---|---|
Luminescence/luciferase, luciferin | 5 nM–100 μM | Cell extract | [47] |
Luminescence/hexokinase, glucose, luminol | 0.30–80/70 | pharmaceuticals, milk, and soils | [48] |
Luminescence/aptamer, [Ru(phen)2(dppz)]2+ | 1–100 nM/1 nM | - | [49] |
Luminescence/aptamer, peroxidase, luminol | 1–10/0.1 | Cancer cell extract | [50] |
Chemiluminescence/luminol-H2O2- HRP-fluorescein | 1–10/0.32 | Cancer cell extract | [50] |
Fluorescence polarization/QD-DNA | 10–350/3.7 | Serum | [51] |
Bioluminescent/luciferase, luciferin, aptamer, Au NPs | 1–600/5 | Cancer cell extract | This work |
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Karimi, E.; Nikkhah, M.; Hosseinkhani, S. Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection. Biosensors 2022, 12, 918. https://doi.org/10.3390/bios12110918
Karimi E, Nikkhah M, Hosseinkhani S. Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection. Biosensors. 2022; 12(11):918. https://doi.org/10.3390/bios12110918
Chicago/Turabian StyleKarimi, Elham, Maryam Nikkhah, and Saman Hosseinkhani. 2022. "Label-Free and Bioluminescence-Based Nano-Biosensor for ATP Detection" Biosensors 12, no. 11: 918. https://doi.org/10.3390/bios12110918