Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor
Abstract
:1. Introduction
2. Results and Discussion
2.1. Selection of Aptamers Specific for DEHP Based on ssDNA Immobilized SELEX
2.2. High-Throughput Sequencing and Sequence Analysis of the Enriched Library
2.3. Establishment of Gold Nanoparticles Colomertric Assay to Identify Active Aptamers
2.4. Characterization of Active Aptamers
2.5. Fabrication of Electrochemical Impedance Spectroscope Aptasensor
2.6. Analytical Application of Sulfhydryl Modified Aptamer 31 in DEHP Detection Using EIS Aptasensor
3. Materials and Methods
3.1. Chemicals and Reagents
3.2. Selection of Aptamers Specific for DEHP Based on ssDNA library immobilized SELEX
3.3. Establishment of a Real-Time Quantitative PCR Method for the Monitoring Selection Process
3.4. Preparation of Secondary Libraries
3.5. High-Throughput Sequencing and Sequence Analysis of the Enriched Library
3.6. Determination of Binding Activity Between Aptamers and DEHP Using a Gold Nanoparticles Colomertric Assay
3.7. Affinity Analysis of Active Aptamers Using LSPR
3.8. Fabrication of EIS Aptasensor
3.9. Specificity and Sensitivity Analysis of Aptamer 31
3.10. Application of the EIS Aptasensor in Spiked Water Samples
4. Conclusions
5. Patents
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Sample Availability: Samples of the compounds are not available from the authors. |
Aptamer | Sequence (5′-3′) | Kd(nM) |
---|---|---|
31 | ACGCATAGGGTGCGACCACATACGCCCCATGTATGTCCCTTGGTTGTGCCCTATGCGT | 2.26 ± 0.06 |
123 | ACGCATAGGGCAACCAGACCAGCCCCATCCCCATGTGACTTCTGTTTGGCCTATGCGT | 5.33 ± 0.01 |
203 | ACGCATAGGGCAAGACAAACTGCGCCATTCAGCATGCTGTTCGGGTTGGCCTATGCGT | 2.68 ± 0.2 |
281 | ACGCATAGGGTGTGCATCAGCAGTACCAACGACGTTGTGGTGTGCTCATCCTATGCGT | 43 ± 0.7 |
Methods | Linear Range | Limit of Detection (LOD) | References |
---|---|---|---|
High-performance liquid chromatography | 50–100,000 ng/mL | / | 5–7 |
High-performance liquid chromatography–mass spectrometry | 0.01–0.1 ng/mL | 1 pg/mL | 8 |
Raman spectroscopy | 0.008–182nM | 3.12 pg/mL | 14 |
Mass spectrometry | 5–1000 ng/mL | 210 pg/mL | 10 |
Enzyme-linked immunosorbent assay | 10−3–103 ng/mL | 4.2 pg/mL | 11 |
Multiresidue detection method based on aptamer | 0.5–30 ng/mL | 3.9 pg/mL | 12 |
An ultrasensitive electrochemical method | 7.629 pg/mL-2 µg/mL | 0.103 pg/mL | This work |
Samples | Spiked Concentration (μg·L−1) | Detected Concentration (μg·L−1) | RSD % | Recovery % |
---|---|---|---|---|
Tap water | 0.03 | 0.031 | 2.74 | 101.77 |
1.95 | 1.71 | 1.44 | 87.37 | |
500 | 462.59 | 1.57 | 92.52 | |
Water from YB | 0.03 | 0.043 | 1.69 | 141.32 |
1.95 | 1.73 | 2.50 | 88.50 | |
500 | 488.44 | 2.54 | 97.69 | |
Water from Qingshan Lake | 0.03 | 0.026 | 2.15 | 85.70 |
1.95 | 1.49 | 2.19 | 76.07 | |
500 | 481.22 | 0.55 | 96.24 |
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Lu, Q.; Liu, X.; Hou, J.; Yuan, Q.; Li, Y.; Chen, S. Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor. Molecules 2020, 25, 747. https://doi.org/10.3390/molecules25030747
Lu Q, Liu X, Hou J, Yuan Q, Li Y, Chen S. Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor. Molecules. 2020; 25(3):747. https://doi.org/10.3390/molecules25030747
Chicago/Turabian StyleLu, Qi, Xixia Liu, Jianjun Hou, Qiuxue Yuan, Yani Li, and Sirui Chen. 2020. "Selection of Aptamers Specific for DEHP Based on ssDNA Library Immobilized SELEX and Development of Electrochemical Impedance Spectroscopy Aptasensor" Molecules 25, no. 3: 747. https://doi.org/10.3390/molecules25030747