An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies
Abstract
:1. Introduction
2. Materials and Methods
2.1. Reagents and Instruments
2.2. Preparation of Gold-Magnetic Nanoparticles
2.3. Preparation and Characterization of Anti-Ricin Phage Displayed Antibody
2.3.1. Preparation of a Large Phage Displayed Single-Chain Fragment Variable (scFv) Antibody Library
2.3.2. Screening of Anti-Ricin Phage Displayed Antibody
2.3.3. Characterization of Anti-Ricin Phage Displayed Antibody Positive Clones by ELISA
2.3.4. DNA Sequencing and Binding Affinity of Anti-Ricin Phage Displayed Antibody
2.4. Preparation of the SPA-Coated Gold-Magnetic Nanoparticles Functionalized Capturing Probes
2.5. Preparation of ECL Probes
2.6. Establishment of the ECL Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibody
2.7. Regeneration of the Electrodes Surface of ECL Immunosensor
2.8. Limit of Detection, Linear Range and Specificity
2.9. Measurement of Simulated Ricin Samples
3. Results
3.1. Preparation of Gold-Magnetic Nanoparticles
3.2. Preparation of Magnetic Ricin-Capturing Probe
3.2.1. Optimal Amount of Immobilized Anti-Ricin Polyclonal Antibody
3.2.2. Qualification of the SPA-Coated Gold-Magnetic Nanoparticles Functionalized Capturing Probe
The Magnetic Features of the SPA-Coated Gold-Magnetic Nanoparticles Functionalized Capturing Probe
Biological Activity of the SPA-Coated Gold-Magnetic Nanoparticles Functionalized Capturing Probe
3.3. The Preparation and Characterization of Anti-Ricin Phage Displayed Antibody
3.3.1. Screening of Anti-Ricin Phage Displayed Antibody
3.3.2. Characterization of Anti-Ricin Phage Displayed Antibody Positive Clones by ELISA
3.3.3. DNA Sequencing and Binding Affinity of Anti-Ricin Phage Displayed Antibody
3.4. The Qualification Feature of ECL Probe
3.4.1. Analysis of UV-Vis Spectrum of ECL Probe
3.4.2. Qualification of the ECL Profile of the ECL Probe
3.5. The Performance of ECL Immunosensor
3.5.1. Linear Range and Limit of Detection
3.5.2. Accuracy and Specificity
3.5.3. Detection of Ricin in Simulated Samples
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Added Amount (µg) | A280nm before | A280nm after | Binding Ratio (%) | Immobilized Amount (µg) |
---|---|---|---|---|
40 | 0.428 ± 0.005 | 0.043 ± 0.003 | 75.0 | 30 |
80 | 0.773 ± 0.007 | 0.106 ± 0.006 | 72.5 | 58 |
160 | 1.077 ± 0.008 | 0.249 ± 0.005 | 63.8 | 102 |
240 | 1.498 ± 0.007 | 0.530 ± 0.008 | 46.3 | 111 |
320 | 1.959 ± 0.009 | 0.931 ± 0.008 | 35.3 | 113 |
400 | 2.443 ± 0.008 | 1.404 ± 0.009 | 29.3 | 117 |
500 | 3.037 ± 0.009 | 1.998 ± 0.008 | 24.0 | 120 |
Added Amount (µg) | A280nm before | A280nm after | Binding Ratio (%) | Immobilized Amount (µg) |
---|---|---|---|---|
40 | 0.428 ± 0.006 | 0.107 ± 0.004 | 90.0 | 36 |
80 | 0.773 ± 0.008 | 0.213 ± 0.006 | 86.3 | 69 |
160 | 1.077 ± 0.007 | 0.390 ± 0.006 | 76.9 | 123 |
240 | 1.498 ± 0.008 | 0.800 ± 0.007 | 64.6 | 155 |
320 | 1.959 ± 0.009 | 1.267 ± 0.008 | 52.5 | 168 |
400 | 2.443 ± 0.008 | 1.720 ± 0.007 | 42.3 | 169 |
500 | 3.037 ± 0.009 | 2.308 ± 0.009 | 34.2 | 171 |
Added Amount (µg) | A280nm before | A280nm after | Binding Ratio (%) | Immobilized Amount (µg) |
---|---|---|---|---|
40 | 0.434 ± 0.008 | 0.043 ± 0.006 | 90.0 | 36 |
80 | 0.795 ± 0.009 | 0.099 ± 0.007 | 87.5 | 70 |
160 | 1.094 ± 0.008 | 0.219 ± 0.008 | 80.0 | 128 |
240 | 1.518 ± 0.009 | 0.411 ± 0.006 | 72.9 | 175 |
320 | 1.923 ± 0.007 | 0.600 ± 0.009 | 68.8 | 220 |
400 | 2.446 ± 0.009 | 0.824 ± 0.008 | 66.3 | 265 |
480 | 2.956 ± 0.010 | 1.318 ± 0.009 | 55.4 | 266 |
560 | 3.550 ± 0.010 | 1.864 ± 0.010 | 47.5 | 266 |
Round | Phage Input (PFU) | Phage Output (PFU) | Recovery Ratio (%) |
---|---|---|---|
1 | 3.3 × 1012 | 6.2 × 105 | 1.9 × 10−7 |
2 | 9.6 × 1011 | 9.7 × 105 | 1.0 × 10−6 |
3 | 1.4 ×1012 | 2.2 × 106 | 1.6 × 10−6 |
Detecting Scheme | Linear Range (μg/L) | Regression Equation | Correlation Coefficient (R) | Limit of Detection (μg/L) |
---|---|---|---|---|
Method 1 | 0.0001~200 | Y = 219.42X + 748.61 | 0.9903 | 0.0001 |
Method 2 | 0.0003~200 | Y = 204.83X + 643.88 | 0.9918 | 0.0003 |
Method 3 | 0.0003~200 | Y = 194.17X + 613.61 | 0.9919 | 0.0003 |
Method 4 | 0.002~500 | Y = 171.42X + 398.51 | 0.9905 | 0.002 |
Method 5 | 0.018~500 | Y = 158.86X + 236.21 | 0.9913 | 0.018 |
Method 6 | 0.25~250 | Y = 0.5415X + 0.6196 | 0.9945 | 0.25 |
Detection Method | Ricin Enrichment Method | Sample Matrix | LOD | Time | Reference |
---|---|---|---|---|---|
Sandwich-type ELISA based on microwave irradiation and heat | Antibody conjugated to 96-well plate | Food Samples | 10 ppb | 2 h | [37] |
ELISA and Luminex fluid array assays | sdAb conjugated to 96-well plate | Buffer | 1 ng/mL and 64 pg/mL | Not reported | [38] |
Lateral flow devices | Antibody conjugated nitrocellulose membrane | Cosmetics | 0.01 μg/mL | Not reported | [39] |
Colloidal immunochromatographic assay | Antibody conjugated nitrocellulose membrane | Buffer | 0.1~50 ng/mL | 10 min | [40] |
Electrochemiluminescence assay | Antibody conjugated to 96-well plate | Buffer | 50 pg/mL | 2.5 h | [41] |
Magnetoelastic sensor | Antibody conjugated to sensor surface | Water, blood and serum | 5 ng/mL | 3.5 h | [42] |
Microring resonator array | sdAb conjugated to microring resonator array | Buffer | 300 pM | 15 min | [43] |
Surface plasmon resonance based on sdAb-QD | sdAb conjugated to SPR chip | Buffer | 1 ng/mL and 0.7 ng/mL | 2~6 min | [44] |
Antibody-sandwich surface plasmon resonance sensor | Antibody conjugated to SPR chip | Buffer | 3 ng/mL | <30 min | [45] |
Liquid-crystal based sensor | Antibody conjugated to liquid crystals supported surfaces | Buffer | 10 μg/mL | 1–2 h | [46] |
Electrochemical aptamer scaffold biosensors | Aptamer conjugated to gold electrode surface | Buffer | 0.3~0.1 nM | Not reported | [47] |
Nanoelectrode array biosensor based on carbon nanofiber | Antibody or aptamer conjugated to the carbon nanofibers chips | Buffer | <1 pM | 4 h | [48] |
DNA aptamer and Raman scattering technique | Aptamer conjugated to magnetic particles | Buffer and beverages | 25 ng/mL | Not reported | [49] |
Immuno-PCR | Antibody conjugated to microtitration plate | Ground beef, milk, and egg | 0.01~0.1 ng/mL | Not reported | [50] |
Real-time fluorescence PCR of nanoparticle-based bio-barcode | Antibody conjugated to magnetic nanoparticle | Buffer | 1 fg/mL | Not reported | [51] |
Nano LC–MS | Lactose-immobilized monolithic spin column | High protein solution | 8 ng/mL | 5 h | [52] |
Object | ECL Intensity (a.u.) | Relative Standard Deviation (%) |
---|---|---|
Ricin | 737.8 ± 13.5 | 1.8 |
Abrin | 5.2 ± 0.5 | 8.6 |
SEB | 5.6 ± 0.5 | 9.8 |
BSA | 5.6 ± 0.5 | 9.8 |
River water | 4.8 ± 0.4 | 9.3 |
Fertilized soil | 5.2 ± 0.4 | 8.6 |
Butter biscuit | 5.2 ± 0.4 | 8.6 |
Whole rabbit blood | 5.6± 0.5 | 9.8 |
PBS buffer | 5.2 ± 0.4 | 8.6 |
Sample | Added (μg/L) | Found (μg/L) | Recovery Ratio (%) | Relative Standard Deviation (%) |
---|---|---|---|---|
River water | 5 | 4.71 ± 0.15 | 94.2 | 3.08 |
Fertilized soil | 5 | 4.58 ± 0.07 | 91.6 | 1.46 |
Butter biscuit | 5 | 4.55 ± 0.14 | 91.1 | 2.97 |
Whole rabbit blood | 5 | 4.54 ± 0.11 | 90.8 | 2.38 |
Sample | Added (μg/L) | Found (μg/L) | Recovery Ratio (%) | Relative Standard Deviation (%) |
---|---|---|---|---|
River water | 5 | 4.61 ± 0.12 | 92.2 | 2.60 |
Fertilized soil | 5 | 5.77 ± 0.11 | 115.4 | 1.91 |
Butter biscuit | 5 | 4.55 ± 0.09 | 91.0 | 1.98 |
Whole rabbit blood | 5 | 5.96 ± 0.14 | 119.2 | 2.35 |
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Mu, X.; Tong, Z.; Huang, Q.; Liu, B.; Liu, Z.; Hao, L.; Dong, H.; Zhang, J.; Gao, C. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies. Sensors 2016, 16, 308. https://doi.org/10.3390/s16030308
Mu X, Tong Z, Huang Q, Liu B, Liu Z, Hao L, Dong H, Zhang J, Gao C. An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies. Sensors. 2016; 16(3):308. https://doi.org/10.3390/s16030308
Chicago/Turabian StyleMu, Xihui, Zhaoyang Tong, Qibin Huang, Bing Liu, Zhiwei Liu, Lanqun Hao, Hua Dong, Jinping Zhang, and Chuan Gao. 2016. "An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies" Sensors 16, no. 3: 308. https://doi.org/10.3390/s16030308
APA StyleMu, X., Tong, Z., Huang, Q., Liu, B., Liu, Z., Hao, L., Dong, H., Zhang, J., & Gao, C. (2016). An Electrochemiluminescence Immunosensor Based on Gold-Magnetic Nanoparticles and Phage Displayed Antibodies. Sensors, 16(3), 308. https://doi.org/10.3390/s16030308