Design of an Affibody-Based Recognition Strategy for Human Epidermal Growth Factor Receptor 2 (HER2) Detection by Electrochemical Biosensors
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
- Buffer A: phosphate-buffered saline (PBS, 0.10 M, pH = 5.0), containing 140 mM NaCl (with and without 0.05% Tween 20);
- Buffer B: phosphate-buffered saline (PBS, 0.10 M, pH = 7.4), containing 0.10 M KCl (with and without 0.05% Tween 20);
- Buffer C: diethanolamine buffer (DEA, 0.10 M, pH = 9.6), containing 1.0 mM MgCl2 and 100 mM KCl (with and without 0.05% Tween 20 and 0.1% BSA);
- Buffer D: Tris buffer (20 mM, pH = 7.4), containing 150 mM NaCl (with and without 0.05% Tween 20).
2.2. Apparatus
2.3. Development of Affibody-Based Assay
2.3.1. Immobilization of the Biotinylated Affibody
2.3.2. Blocking Step
2.3.3. Reaction with HER2 Protein
2.3.4. Binding with the Biotinylated Affibody and Streptavidin-Alkaline Phosphatase Enzyme
2.3.5. Electrochemical Measurements
2.4. Development of Antibody/Affibody Based Assay
2.4.1. Immobilization of the Antibody
2.4.2. Blocking Step
2.4.3. Reaction with HER2 Protein and Biotinylated Affibody, Labeling with Streptavidin-Alkaline Phosphatase and Electrochemical Measurements
2.5. Development of Affibody/Antibody-Based Assay
Binding with Biotinylated Antibody and Streptavidin-Alkaline Phosphatase Enzyme
2.6. Analysis of Serum Samples
3. Results and Discussion
3.1. Optimization of Experimental Parameters
3.2. Sensitivity and Reproducibility
3.3. Analysis of Serum Samples
4. Conclusions
Supplementary Materials
Author Contributions
Conflicts of Interest
References
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Assay Step | Parameter | Current Difference (μA) (ΔI = IHER2 – IBlank) | %RSD | |
---|---|---|---|---|
a | Biot-Af concentration | 1 μg/mL | 1.5 | 8 |
5 μg/mL | 3.3 | 7 | ||
10 μg/mL | 3.2 | 10 | ||
Biot-Af incubation time | 120 min | 1.7 | 7 | |
240 min | 2.0 | 10 | ||
o.n. | 3.3 | 7 | ||
b | Biotin incubation time | 30 min | 1.2 | 8 |
60 min | 2.2 | 9 | ||
90 min | 3.3 | 7 | ||
c | HER2 incubation time | 10 min | 1.3 | 6 |
20 min | 3.3 | 7 | ||
60 min | 3.0 | 9 | ||
d | Biot-Af concentration | 1 μg/mL | 1.2 | 8 |
5 μg/mL | 3.3 | 7 | ||
10 μg/mL | 3.5 | 10 | ||
Biot-Af incubation time | 30 min | 1.6 | 7 | |
45 min | 3.3 | 7 | ||
60 min | 2.9 | 9 |
HER2 Spiked (ng/mL) | HER2 Found (ng/mL) | Recovery (%) | Bias (%) | %RSD |
---|---|---|---|---|
5 | 5.5 | 110 | 10 | 12 |
10 | 11 | 108 | 8 | 11 |
15 | 15 | 97 | −3 | 14 |
20 | 19 | 95 | −5 | 13 |
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Ilkhani, H.; Ravalli, A.; Marrazza, G. Design of an Affibody-Based Recognition Strategy for Human Epidermal Growth Factor Receptor 2 (HER2) Detection by Electrochemical Biosensors. Chemosensors 2016, 4, 23. https://doi.org/10.3390/chemosensors4040023
Ilkhani H, Ravalli A, Marrazza G. Design of an Affibody-Based Recognition Strategy for Human Epidermal Growth Factor Receptor 2 (HER2) Detection by Electrochemical Biosensors. Chemosensors. 2016; 4(4):23. https://doi.org/10.3390/chemosensors4040023
Chicago/Turabian StyleIlkhani, Hoda, Andrea Ravalli, and Giovanna Marrazza. 2016. "Design of an Affibody-Based Recognition Strategy for Human Epidermal Growth Factor Receptor 2 (HER2) Detection by Electrochemical Biosensors" Chemosensors 4, no. 4: 23. https://doi.org/10.3390/chemosensors4040023