Molecular tools for the rapid and cost-effective detection of small molecules
- Autores: Xhensila Shkembi
- Directores de la Tesis: Ciara K. O'Sullivan (dir. tes.), Vasoula Skouridou (codir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2021
- Idioma: español
- Tribunal Calificador de la Tesis: Arben Merkoçi (presid.), Mònica Campàs i Homs (secret.), Maria C. DeRosa (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: TDX
- Resumen
The interest in detecting low molecular weight targets has increased in recent years. Monitoring the level of biologically active small molecules such as antibiotics, toxins, molecular markers, metal ions, drugs and steroids among others is very important in the biomedical field, environmental monitoring and food safety. Sensitive, specific, affordable and preferable portable analytical assays and biosensors suitable for the detection of these molecules are therefore required to ensure public health. In this thesis, two different types of small molecules were targeted: the marine toxin tetrodotoxin and the anabolic steroid nandrolone.
Tetrodotoxin (TTX) is a paralytic marine neurotoxin causing seafood poisoning after the ingestion of contaminated marine food such as puffer fish and shellfish. Its paralytic toxic effects derive from its selective binding to voltage-gated sodium channels and ultimately interfering with neural transmission. Symptoms of TTX intoxication include numbness sensation in the mouth, headache, vomiting, muscle weakness, and even fatal respiratory or heart failure. Puffer fish poisoning is typical of warm waters and was regarded as a problem confined to Asian countries, but in recent years, TTX has been reported and detected in seafood in many different European countries including Spain, Greece, United Kingdom, France and Italy. The high toxicity and the increased incidence of TTX intoxication requires fast and cost-effective detection techniques.
The second target studied is nandrolone. This is an androgenic anabolic steroid (AAS) functioning as a growth promoting agent which helps to gain muscle weight. Its AAS properties have let to its exploitation as a doping agent in sports and horse racing, whereas it is also used as an animal feed additive. On the other hand, several studies report the presence of nandrolone in dietary supplements as a cross-contaminant and consumption of such supplements could lead to accidental doping. Adverse side effects are associated with nandrolone accumulation in the body such as endocrine, cardiovascular, skin and psychiatric disorders. It is therefore evident that monitoring the presence of nandrolone in human and animal biological fluids, meat products and nutritional supplements is essential to protect public health and discourage doping practices in sports.
For both types of molecules, liquid and gas chromatography-mass spectroscopy is routinely used for laboratory-based analysis of field samples. Competitive immunoassays have also been developed and are available in the market for their detection. Aptamers are biorecognition molecules considered as alternative to antibodies which are suitable for the detection of any type of target and have great potential in analytical applications. They are artificial synthetic nucleic acids (RNA/DNA) that bind specifically to their target, and they are selected through an in vitro iterative process called Systematic Evolution of Ligands by Exponential enrichment (SELEX). The development of aptamers for small molecules is a challenging task, considering the low availability of functional groups in their structures. In this work, novel aptamers binding to TTX and Nandrolone were developed and exploited for the detection of the target molecules in different bioassays and biosensors.
For TTX aptamers identification, a variation of SELEX suitable for small molecules termed Capture-SELEX was used, while a classical SELEX process with the target immobilized on sepharose resing was used for nandrolone. Both selections were combined with high-throughput Next Generation Sequencing to identify aptamer candidates and their binding properties were characterized with various assays. Finally, the best-performing aptamers were used for the development of different assays and detection of the target molecules. For TTX, a highly sensitive antibody-aptamer sandwich assay was developed in a microtiter plate format enabling the detection of TTX in buffer and in TTX-containing puffer fish extracts. A portable lateral flow dipstick assay was also successfully developed using the same sandwich antibody-aptamer format. For Nandrolone, a label-free aptamer-based colorimetric assay was developed based on gold nanoparticles and their aggregation in the presence of target molecules after salt addition.
In the thesis, the general objectives and subobjectives of this work are discussed.
Chapter 1 includes a detailed introduction of the state of art of the research area. It provides a brief information about aptamers and their methods of selection against different types of small molecules by using different types of selection. Moreover, a general view of different types of aptasensor used for small molecules detection is provided.
The detailed selection process of the TTX aptamers isolation is detailed in Chapter 2. Capture SELEX was implemented for immobilizing the ssDNA library on the surface of streptavidin magnetic beads and TTX target is free in the solution throughout selection process. The affinity of the selected aptamers was confirmed by two different methodologies, APAA and Bead-ELAA. Finally, a hybrid antibody-aptamer sandwich assay was developed for TTX detection in puffer fish extracts.
Chapter 3 describes the development of a portable, highly specific and sensitive lateral flow dipstick assay TTX detection. The assay was succesfully applied for the detection of TTX in contaminated puffer fish extractse from different tissues (gonads, muscle, skin and liver).
In Chapter 4, the selection performed for the identification of nandrolone aptamers is detailed and the characterization of the binding properties of the aptamer candidates. Finally, an easy and rapid colorimetric assay was developed for nandrolone detection exploiting gold nanoparticles. Adsorption of unmodified aptamer on the surface of gold nanoparticles prevents their aggregation after salt addition. In the presence of nandrolone, displacement of the aptamer to the solution phase to enable target binding results in deprotection of the gold nanoparticles and their aggregation after salt addition, with a concomitant red-to-blue color change.
