Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry
Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-06-01
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| Series: | Biosensors |
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| Online Access: | https://www.mdpi.com/2079-6374/13/6/660 |
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| author | Richa Sharma Monali Mukherjee Praveena Bhatt K. S. M. S. Raghavarao |
| author_facet | Richa Sharma Monali Mukherjee Praveena Bhatt K. S. M. S. Raghavarao |
| author_sort | Richa Sharma |
| collection | DOAJ |
| description | Aptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3′ end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL<sup>−1</sup>) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol. |
| first_indexed | 2024-03-11T02:41:34Z |
| format | Article |
| id | doaj.art-2fb7b78ecb9e473688074026b9ddefff |
| institution | Directory Open Access Journal |
| issn | 2079-6374 |
| language | English |
| last_indexed | 2024-03-11T02:41:34Z |
| publishDate | 2023-06-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Biosensors |
| spelling | doaj.art-2fb7b78ecb9e473688074026b9ddefff2023-11-18T09:33:20ZengMDPI AGBiosensors2079-63742023-06-0113666010.3390/bios13060660Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer InterferometryRicha Sharma0Monali Mukherjee1Praveena Bhatt2K. S. M. S. Raghavarao3Department of Food Engineering, CSIR-Central Food Technological Research Institute (CFTRI), Mysore 570020, IndiaAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, IndiaAcademy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, IndiaDepartment of Food Engineering, CSIR-Central Food Technological Research Institute (CFTRI), Mysore 570020, IndiaAptamers are an excellent choice for the selective detection of small molecules. However, the previously reported aptamer for chloramphenicol suffers from low affinity, probably as a result of steric hindrance due to its bulky nature (80 nucleotides) leading to lower sensitivity in analytical assays. The present work was aimed at improving this binding affinity by truncating the aptamer without compromising its stability and three-dimensional folding. Shorter aptamer sequences were designed by systematically removing bases from each or both ends of the original aptamer. Thermodynamic factors were evaluated computationally to provide insight into the stability and folding patterns of the modified aptamers. Binding affinities were evaluated using bio-layer interferometry. Among the eleven sequences generated, one aptamer was selected based on its low dissociation constant, length, and regression of model fitting with association and dissociation curves. The dissociation constant could be lowered by 86.93% by truncating 30 bases from the 3′ end of the previously reported aptamer. The selected aptamer was used for the detection of chloramphenicol in honey samples, based on a visible color change upon the aggregation of gold nanospheres caused by aptamer desorption. The detection limit could be reduced 32.87 times (1.673 pg mL<sup>−1</sup>) using the modified length aptamer, indicating its improved affinity as well as its suitability in real-sample analysis for the ultrasensitive detection of chloramphenicol.https://www.mdpi.com/2079-6374/13/6/660antibioticsaffinitybiosensinginterferometrynanoparticles |
| spellingShingle | Richa Sharma Monali Mukherjee Praveena Bhatt K. S. M. S. Raghavarao Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry Biosensors antibiotics affinity biosensing interferometry nanoparticles |
| title | Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry |
| title_full | Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry |
| title_fullStr | Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry |
| title_full_unstemmed | Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry |
| title_short | Rational Truncation of Aptamer for Ultrasensitive Aptasensing of Chloramphenicol: Studies Using Bio-Layer Interferometry |
| title_sort | rational truncation of aptamer for ultrasensitive aptasensing of chloramphenicol studies using bio layer interferometry |
| topic | antibiotics affinity biosensing interferometry nanoparticles |
| url | https://www.mdpi.com/2079-6374/13/6/660 |
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