Application of dna binding protein tags as means for defined co-immobilization in biosensors
- Autores: Gülsen Betül Aktas
- Directores de la Tesis: Lluís Masip Vernis (dir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Aart Van Amerongen (presid.), Mònica Campàs i Homs (secret.), María Isabel Pividori (voc.)
- Programa de doctorado: Programa de Doctorado en Nanociencia, Materiales e Ingeniería Química por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: hdl.handle.net
- Resumen
Proteins are the primary functional agents that play essential roles in life processes. Since their discovery, proteins had a big impact in the different research areas especially as the building blocks of biosensors. The field of the biosensors has been hailed as the detection to many diseases in health industry. The protein immobilization mediated the improvement of biosensors from bottom to top; surface immobilization to detection element.
There have been different methods established to immobilize the proteins on the surfaces of the biosensors; physical adsorption, covalent linking and the bioaffinity interactions. Each of them has advantages and disadvantages but none of the established method provides a controlled co-immobilization of proteins. Therefore, a robust method for the controlled protein co-immobilization in biosensor is necessary to improve the biosensor sensing system and the detection of the several diseases.
The objective of this doctoral thesis is to explore the use of DNA binding proteins in biosensors mainly as universal detection molecules. The specificity and high affinity that these proteins exhibit for their respective double stranded DNA (dsDNA) sequence was thus exploited in order to develop a protein immobilization technique that allows the controlled co-immobilization of several proteins. The proposed system is based on the utilization of these DNA binding proteins as fusion tags for the target proteins to be immobilized and the use of dsDNA as the template to direct the protein co-immobilization by specific positioning of the target DNA sequences of the DNA binding proteins. The preparation and characterization of two novel enzyme-DNA binding protein conjugates and their use for detection and signal amplification in biosensors is described.
The thesis is divided in six chapters:
Chapter 1 contains a general introduction and literature review, which includes a brief information on controlled co-immobilization at the molecular scale of several proteins. In addition, an overview of DNA binding proteins, their preparation and conjugation are presented in this chapter.
The first application of the proposed system is described in Chapter 2. A novel DNA sensing platform is designed and it is based on scCro DNA binding protein tag-HRP enzyme conjugate and a hybrid single stranded DNA-double stranded DNA (ssDNA-dsDNA) detection probe for the model ssDNA target high-risk human papillomavirus (HPV16). The number of HRP molecules associated with each target DNA molecule is controlled by the number of DNA binding sites on the hybrid ssDNA-dsDNA detection probe and the ssDNA target is detected using an enzyme linked oligonucleotide assay with improved detection signal. The described platform serves as a proof of concept for the protein co-immobilization system. It is universal since the HRP-scCro protein conjugate can be used for the detection of any ssDNA target by changing only the ssDNA part of the hybrid ssDNA-dsDNA detection probe.
An alternative design of the protein co-immobilization system is demonstrated in Chapter 3. Two different DNA binding protein-enzyme conjugates are used together with a simple DNA nanostructure for the detection of ssDNA target, using again the HPV16 as model target. The DNA nanostructure, which is the hybrid ssDNA-dsDNA detection probe, performs two functions: its ssDNA part is complementary to the target sequence and serves for target recognition whereas the dsDNA part provides signal generation through the specific binding sites for the two different DNA binding protein tags of the enzyme conjugates. The two enzyme-DNA binding protein conjugates GOx-dHP and HRP-scCro form an enzymatic cascade which results in signal generation that does not require the external addition of hydrogen peroxide. The effect of enzyme ratio on the obtained signal is also examined by using different designs of the detection probe.
The possibility of using the protein co-immobilization system for non-nucleic acid targets such as proteins is demonstrated in Chapter 4 by using DNA aptamers. This novel strategy requires a pair of aptamers (dual aptamers) which are modified in a way that each aptamer contains the specific dsDNA binding site for each of the GOx-dHP and HRP-scCro enzyme conjugates. One aptamer serves for target capturing, whereas the second one for detection in a sandwich-type aptasensor, where signal is generated as a result of the simultaneous binding of each enzyme conjugate to each modified aptamer sequence This system was explored for thrombin detection and the limit of detection achieved was comparable to other systems reported in the literature. The possible use of this detection system in low resource settings at the point of care is also demonstrated by a preliminary paper-based, lateral flow assay.
A more detailed study on the use of DNA binding proteins in lateral flow assays is provided in Chapter 5. Here, a novel direct detection of the target sequence after PCR amplification utilizing the report system based on DNA binding proteins and specific dsDNA sequences in a lateral flow configuration is described. Direct detection of the target dsDNA PCR product without the need to generate ssDNA after the PCR step is achieved by incorporating the DNA binding sequence recognized by DNA binding protein in the reverse PCR primer sequence. The forward primer is also modified with a label (Alexa Fluor 488) to enable target amplicon capture on the lateral flow strip by a specific monoclonal antibody binding the Alexa Fluor 488 label. Detection is accomplished using three different approaches: with a scCro/carbon nanoparticle conjugate, leading to carbon black color, HRP-scCro enzyme conjugate (HRP precipitating red dye colur) and HRP-scCro/carbon nanoparticle conjugate (carbon black plus HRP precipitating red dye colour). The system is validated for the detection of Shiga-toxin producing Escherichia coli and low limits of detection are achieved.
This thesis concludes in Chapter 6 with a summary and significance of the completed work and the future perspectives of this research.
