Exhaled breath analysis for non-invasive diagnosis of tropical diseases
- Autores: Tesfalem Geremariam Welearegay
- Directores de la Tesis: Ionescu Radu (dir. tes.), Eduard Llobet Valero (dir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2019
- Idioma: español
- Tribunal Calificador de la Tesis: X. Vilanova (presid.), José Santiago Torrecilla Velasco (secret.), Boris Mizaikoff (voc.)
- Programa de doctorado: Programa de Doctorado en Tecnologías para Nanosistemas, Bioingeniería y Energía por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: TDX
- Resumen
Neglected Tropical Diseases (NTDs) belong to the group of infectious diseases. They are endemic in most parts of the world, affecting more than one billion people worldwide, especially low income populations from developing regions. The infection to humans is characterized by a chronic and prolonged asymptomatic incubation period without noticeable symptoms of the disease, which delays the prescription of a suitable and timely medical treatment. The prevention, diagnosis and control of these diseases still remain an unsolved medical challenge.
In line with the search for non-invasive disease diagnostic tools, exhaled breath analysis has brought a great clinical potential for not only disease detection capability but also disease classification as well as therapeutic monitoring of patient’s condition. The fact that exhaled breath comprises certain volatile organic compounds (VOCs) with relatively low molecular weight, expresses the immediate changes in pathological and metabolic process disorders that are correlated to the existence of a specific disease in the human body; as compared with the healthy state.
Hence, despite the current practice of breath analysis using complex analytical techniques; cross-reactive nanomaterial based on chemical sensor array combined with pattern recognition present as a new frontier of non-invasive, rapid and potentially inexpensive diagnostic approach. Such chemical sensors array are based on monodispersed metallic nanoparticles (MNPs) tailored with various molecular organic ligands that can provide a sorption site for the multicomponent VOCs; up on which their interaction affect the chemical and electronic property of the MNPs-ligand nanoassemblies. Although these organic ligand capped metal nanoparticles exhibit remarkable progress for different diseases diagnosis, there still remain challenges in the fabrication and functionalization of these sensing entities and have never been applied in the diagnosis of infectious diseases.
The aim of the PhD thesis was thus twofold: on one hand, a novel technique for the fabrication of ultrapure metal nanoparticles were introduced, while on the other hand it revealed the diagnostic potential of chemical gas sensors based on ultrapure ligand-capped metal nanoparticles for the diagnosis of three different neglected tropical diseases (Dengue, Leishmaniasis and Echinococcosis), whose sensing films were selected based on their affinity to the breath biomarkers of these diseases that was identified through analytical studies.
The first goal of the thesis was to design, fabricate and characterize chemical gas sensors based on ultrapure ligand-capped metal nanoparticles. For this end, it has shown that the Advanced Gas Deposition technique employed in this thesis work provided the synthesis of ultrapure monodispersed metal nanoparticles (MNPs) from the evaporation of a pure metal precursor in vacuum. I focused on the synthesis of three metal nanoparticles (AuNPs, PtNPs and CuNPs), however the novel technique that I introduced in my thesis can be easily extended to practically any MNPs synthesis using the proper metal source and adjusting the synthesis parameters. MNPs capping with organic ligands was achieved by dip-coating the substrates in a solution formed in high purity reagents, which led to the formation of self-assembled nanoparticles-organic monolayers with a network-like structure. The MNPs-ligand nanoassemblies presented hybrid characteristics, where the nanoparticles served for electrical conduction, while the capping organic molecules promoted electron tunneling through the MNPs. The electrical characterization of the AuNPs and PtNPs nanoassemblies led to the first ever observation of Schottky diodes fabricated from nanomaterials based on metal nanoparticles. Preliminary sensing measurements showed the good potential of the novel chemical gas sensors that I produced for breath VOCs detection.
The second goal of the thesis, inspired by the fact that these hybrid nanoassemblies and their diverse functionalitywas, was to develop a non-invasive, easy-to-use and patient-friendly methodology for rapid diagnosis of neglected tropical diseases. For this end, I investigated diseases diagnosis via exhaled breath samples analyses, which are easy to obtain and present no discomfort or risk for patients’ health. In my thesis I focused on three different types of neglected tropical diseases (Dengue, Leishmaniasis and Echinococcosis) caused by three different pathogens (viral, helminthic and protozoan infections, respectively). Breath samples were collected from patients and controls in Colombia (Dengue), Tunisia (Cutaneous Leishmaniasis and Cystic Echinococcosis) and Poland (Alveolar Echinococcosis) with a simple to use Bio-VOCTM breath sampler, and stored in Tenax TA sorbent tubes before analysis.
Analytical studies performed with a GC/Q-TOF equipment (state-of-the-art in analytical chemistry, which was employed for the first time for breath samples analysis in my thesis) identified six putative biomarkers for Dengue (three aromatic compounds, two esters and one alkene), nine putative biomarkers for Cutaneous Leishmaniasis (one ester, two alcohols, one ketone and five alkanes), two putative biomarkers for Cystic Echinococcosis (one alkene and one acid), and seven putative biomarkers for Alveolar Echinococcosis (all alkanes). Importantly, although Cystic Echinococcosis and Alveolar Echinococcosis are from the same family of Echinococcus, the putative biomarkers found for CE and AE were totally different, indicating that the pathogenesis of CE and AE infections and the changes that they produce in the body chemistry appear to be completely different. Classification models built with the identified biomarkers yielded fair to good discrimination accuracies: 84.4% for Dengue diagnosis, 80% for Cutaneous Leishmaniasis diagnosis, 65.5% for Cystic Echinococcosis diagnosis, and 80.9% for Alveolar Echinococcosis diagnosis.
Based on these biomarkers, I selected several chemiresistive gas sensors based on the ultrapure ligand-capped metal nanoparticles that I produced in the first part of my thesis, targeting to detect the breath volatiles patterns of the studied neglected tropical diseases. Although the sensors are not sensitive to a specific volatile, but they respond to the collective exhaled VOCs pattern that comprises a range of compounds with concentrations under the limit of detection of the GC/Q-TOF equipment (and therefore not detected in the analytical studies), the putative biomarkers that I identified did however form a sufficient basis for a rational design of the sensors arrays for each disease.
Classification models built with selected features extracted from sensors responses to the exhaled breath samples of the patients and controls yielded diagnostic accuracies that ranged from 100% for Dengue and Cystic Echinococcosis, to 98.2% for Cutaneous Leishmaniasis and 92.9% for Alveolar Echinococcosis, while the two dominant forms of Human Echinococcal infection (Cystic Echinococcosis and Alveolar Echinococcosis) were discriminated with 92.1% accuracy.
Remarkably, CuNPs-based nanoassemblies yielded higher diagnostic accuracy than AuNPs sensors, which up to date have been by far the most used sensing MNPs materials in breath detection. The CuNPs sensors have higher exposure ratio of the organic functionality to the breath volatiles because of their much smaller particles size (~4 nm) as compared with AuNPs (~10 nm), which is of utmost importance for chemiresistive sensors. Moreover, in comparison with the metal nanoparticles sensors used in previous studies, which employ chemical precursors that often leave traces of residual compounds, the active materials of the sensors used in my thesis were synthesized from a pure metal evaporated in vacuum. With this technique, ultrapure nanoparticles are produced, which ensures reproducible sensors responses with no influence of confounding reactions with synthesis residuals, which is crucial in chemical sensor technology.
Finally, it is worth mentioning that the sensing methodology that I introduced in my thesis for neglected tropical diseases diagnosis is simple to use and interpret, and is amenable for use in both specialist and non-specialist settings. Therefore, it could provide a powerful tool for the facile, rapid and non-invasive diagnosis of NTDs even at an early stage when metabolic changes are already produced, and would be especially important in regions lacking specialized healthcare diagnostic facilities.
