Oligonucleotide Based-Biosensors for Label-Free Electrochemical Protein and DNA Detection.
In the last years, DNA arrays have attracted increasing attention among medical diagnosis and analytical chemists. The broad range of application that has been found for DNA arrays makes them an important analytical tool. DNA arrays are relevant for the diagnosis of genetic diseases, detection of in...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2006 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/8542 |
| Acceso en línea: | http://www.tdx.cat/TDX-0703107-095717 http://hdl.handle.net/10803/8542 |
| Access Level: | acceso abierto |
| Palabra clave: | electroquímica aptamer DNA Biosensor 517 543 577 62 |
| Sumario: | In the last years, DNA arrays have attracted increasing attention among medical diagnosis and analytical chemists. The broad range of application that has been found for DNA arrays makes them an important analytical tool. DNA arrays are relevant for the diagnosis of genetic diseases, detection of infectious agents, study of genetic predisposition, development of a personalised medicine, detection of differential genetic expression, forensic science, drug screening, food safety and environmental monitoring.<br/>Despite the great promise of DNA arrays in health care and their success in medical and biological research, the technology is still far away from the daily use in the clinic and even more far away from their implementation in home-diagnosis such as glucose biosensors. <br/>Their principal problems are the high cost and difficulty of use, because it is required costly laboratory instruments and biology knowledge for the labelling of the DNA prior to the sample injection into the array.<br/>On the other hand, the requirements that a biosensor should include are to be easy-to use so that it do not need the previous label of the sample and the addition of reagents. It should give a sensitive response in short time, and it should also include cheap generic multi-analyte detection.<br/>The work carried out in this thesis describes new concepts of electrochemical biosensoric platforms based on oligonucleotides for detection of label-free DNA and protein, which include these requirements.<br/>Preliminary experiments of direct DNA electrochemical detection of labelled ssDNA were performed to establish a protocol of DNA immobilisation, hybridisation and detection colourimetrically and electrochemically. DNA real samples and multi-analite detection on an array developed by biocopatible photolithography were used.<br/>To avoid the analyte labelling to develop an easy to use and low cost device, a label-free electrochemical displacement of DNA sensor was described. The method of detection by displacement requires the pre-hybridisation of the capture probe immobilised on the electrode surface with a sub-optimum mutated oligonucleotide labelled with a redox molecule. Due to the higher affinity of the target that is fully complementary to the capture probe, the sub-optimum label can be displaced when the complementary target is introduced in the system. The decrease of the signal would verify the presence of the target and should be proportional to its concentration. <br/>Sub-optimum hybridisation displacement detection was demonstrated colourimetrically and electrochemically with a sub-optimum mutated oligonucleotide labelled with horseradish peroxidase (HRP), and a ferrocene sub-optimum mutated oligonucleotide was also detected electrochemically, which do not required the addition of reagents for its detection.<br/>Furthermore different strategies to develop an electrochemical oligonucleotide (aptamer) based sensor for reagentless and label-free protein detection was carried out. The most sensitive aptasensor achieved 30 fM of detection limit in just 5 minutes. |
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