Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Synthesis of Lys-PMNPs
2.2.1. Synthesis of Magnetite NPs
2.2.2. Synthesis of Fe3O4@SiO2 Nanospheres
2.2.3. Synthesis of Amine-Modified Fe3O4 NPs (Fe3O4@SiO2-NH2)
2.2.4. Coupling of the RAFT Agent
2.2.5. Preparation of the Polymer Shell
2.3. Characterization
2.4. Equilibrium Binding Measurement of Lys-PMNPs in Buffer
2.5. Release of Lysozyme from the Lys-PMNPs
2.6. Measurement of Selectivity
2.7. Binding and Release of Lys-PMNPs in Urine
2.8. HPLC Analysis
2.9. Measurement of Urine Samples with Micrococcus lysodeikticus Assay
3. Results and Discussion
3.1. Preparation and Characterization of Poly(N-isopropylacrylamide-co-N-tert-butylacrylamide co-acrylic acid) Coated Magnetic Nanoparticles (Lys-PMNPs)
3.2. Effect of Time on the Binding of Lys-PMNPs
3.3. Effect of Buffer Ionic Strength and pH on the Protein Binding of Lys-PMNPs
3.4. Adsorption Isotherms
3.5. Selectivity towards Different Proteins
3.6. Thermally Modulated Binding and Release of Lysozyme
3.7. Development of a Sample Cleanup Procedure for the Measurement of Lysozyme in Clinical Urine Samples
3.8. Method Validation
3.9. Comparison to a Reference Method by the Measurement of Unknown Samples
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Acknowledgments
Conflicts of Interest
References
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Binding Step | Release Step | ||||||
---|---|---|---|---|---|---|---|
PB Concentration [mM] | pH | Phase Ratio [mg/mL] | PB Concentration [mM] | pH | Additive | Phase Ratio [mg/mL] | Recovery in the Release Step [%] |
10 | 8.6 | 1.6 | 10 | 8.4 | - | 10.67 | 31.2 |
50 | 7 | - | 10.67 | 70.0 | |||
10 | 8.4 | 0.2 M NaCl | 10.67 | 81.2 | |||
50 | 7 | 0.2 M NaCl | 10.67 | 90.6 | |||
50 | 7 | - | 1.6 | 104.2 |
Urine Concentration [µg/mL] | Nominal Sample Concentration [µg/mL] | Measured Concentration [µg/mL] | Precision [%] | Accuracy [%] |
---|---|---|---|---|
25 | 2.5 | 2.94 | 3.44 | 117.6 |
100 | 10 | 9.35 | 8.20 | 93.5 |
250 | 25 | 25.8 | 4.25 | 103.0 |
1000 | 100 | 89.9 | 2.24 | 89.9 |
Urine Concentration [µg/mL] | Nominal Sample Concentration [µg/mL] | Measured Concentration [µg/mL] | Precision [%] | Accuracy [%] |
---|---|---|---|---|
25 | 2.5 | 2.774 | 1.08 | 111.0 |
100 | 10 | 9.442 | 3.21 | 94.4 |
250 | 25 | 24.81 | 5.04 | 99.2 |
1000 | 100 | 88.80 | 4.40 | 88.8 |
2000 | 200 | 128.8 | 2.25 | 64.4 |
Urine Concentration [µg/mL] | Standard Solution Concentration [µg/mL] | Recovery [%] | RSD [%] |
---|---|---|---|
100 | 10 | 91.7 ± 3.8 | 4.19 |
250 | 25 | 103.1 ± 3.9 | 3.80 |
1000 | 100 | 87.5 ± 9.4 | 10.81 |
2000 | 200 | 62.3 ± 6.3 | 10.14 |
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Ahmed, M.A.; Erdőssy, J.; Horvath, V. Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples. Nanomaterials 2021, 11, 3015. https://doi.org/10.3390/nano11113015
Ahmed MA, Erdőssy J, Horvath V. Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples. Nanomaterials. 2021; 11(11):3015. https://doi.org/10.3390/nano11113015
Chicago/Turabian StyleAhmed, Marwa A., Júlia Erdőssy, and Viola Horvath. 2021. "Temperature-Responsive Magnetic Nanoparticles for Bioanalysis of Lysozyme in Urine Samples" Nanomaterials 11, no. 11: 3015. https://doi.org/10.3390/nano11113015