Malaria remains the most important mosquito-borne infectious disease worldwide, with 3.5 billion people still at risk, 228 million cases and 405 000 deaths in 2018. The disease is caused by parasites belonging to the genus of Plasmodium, which infect red blood cells by feeding on haemoglobin and transforming it into hemozoin nanocrystals. Despite the World Health Organization recommends “prompt parasite-based diagnosis in all patients suspected of malaria before treatment is administrated, either by microscopy or malaria rapid diagnostic test (RDTs)”, the quality of microscopy-based diagnosis is frequently inadequate while the reliability of RTDs based on antigens detection is still affected by a sizable number of false negative/positive results. This master’s thesis project is about the optimization and characterization of a lab-on-a-chip pan-malaria rapid diagnostic test, that, based on the paramagnetic properties of hemozoin nanocrystals, combines magnetophoretic separation and electrical impedance detection. The presented diagnostic test, exploiting the competition between gravity and magnetic forces, relies on the local capture of infected red blood cells and hemozoin crystals on micromagnetic concentrators and subsequent detection by electrical measurement. The long-range attraction is provided by external NdFeB permanent magnets, while a concentration on the electrodes is performed by Ni pillars fabricated in the chip. Finally, because of the presence of particles within a solution changes its conductivity, the amount of hemozoin crystals and infected red blood cells can be quantified through an impedance variation detection. During this thesis work, different activities have been carried out to characterize and optimize a diagnostic platform previously developed. First, a solid and reliable analysis code has been realized and used during a preclinical validation study carried out in Cameroon. Then, other important characterization experiments have been performed to demonstrate a limit of detection (LoD) of 10parasites·µl^(-1) and a measurement range from the LoD up to 10^(5)parasites·µl^(-1). During the thesis, the first follow up monitoring has also been carried out. Later, a new microchip with interpenetrated sensitive regions has been designed, fabricated and characterized in order to remove the spurious drift signal affecting the previous chip. This thesis work was part of the TMek project. The experimental activities have been performed in PoliFAB, the micro and nanotechnology centre of “Politecnico di Milano”, under the direct supervision of Prof. Giorgio Ferrari and Prof. Riccardo Bertacco, head of the Nanomagnetism for Biology and Spintronics group of the Department of Physics, and in “Luigi Sacco” hospital in Milan. The detection system has been developed in collaboration with the "I3N" group of “Politecnico di Milano”, directed by Prof. Marco Sampietro and Prof. Giorgio Ferrari, that designed, fabricated and characterized the electronics of the device. The blood samples used in the calibration tests have been provided and treated by "µBS Lab" of "Politecnico di Milano”, led by Prof. Gianfranco B. Fiore, while the preclinical validation campaign has been carried out in the diagnostic laboratory of Hôpital Saint Luc of Mbalmayo in Cameroon.
La malaria con 3.5 miliardi di persone ancora a rischio, 228 milioni di casi e 405 000 morti nel 2018, rimane la più importante malattia al mondo causata da puntura di zanzara. La malattia è causata dai parassiti del genere Plasmodium, che infettano i globuli rossi nutrendosi dell' emoglobina e trasformandola in nanocristalli di emozoina. Nonostante l' Organizzazione Mondiale della Sanità raccomandi una "pronta diagnosi, o con microscopio o usando test diagnostici rapidi (RDT), in tutti i pazienti sospetti di malaria prima che venga loro somministrato un trattamento", la qualità della diagnosi con microscopio è frequentemente inadeguata, mentre l'affidabilità degli RDT basati sull' individuazione di antigeni è ancora affetta da un considerevole numero di falsi positivi e falsi negativi. Questo progetto di tesi magistrale tratta l'ottimizzazione e la caratterizzazione di un lab-on-a-chip test diagnostico rapido pan-malarico che, basandosi sulle proprietà magnetiche dell'emozoina, combina la separazione magnetoforetica e la tecnica di misura impedenziometrica. Il test diagnostico presentato, sfruttando la competizione tra la gravità e le forze magnetiche, si basa sulla cattura locale dei globuli rossi infetti e dei cristalli di emozoina su concentratori micromagnetici e la seguente misura elettrica. L'attrazione su lungo raggio è data da magneti esterni permanenti di NdFeB, mentre una concentrazione sugli elettrodi è eseguita da strutture di Ni fabbricate nel chip. Infine, a causa del cambio di conduttività in una soluzione data la presenza di particelle, la quantità di cristalli di emozoina e globuli rossi infetti può essere valutata attraverso una misura impedenziometrica. Durante questo lavoro di tesi, sono state eseguite diverse attività in modo da caratterizzare e ottimizzare la piattaforma diagnostica precedente. In primo luogo è stato scritto un codice di analisi robusto e affidabile, usato poi durante la validazione clinica in Camerun. Dopodiché, altri importanti esperimenti sono stati condotti volti a dimostrare il valore minimo di parassitemia misurabile (LoD) di 10parassiti·µl^(-1) e una curva di calibrazione dal LoD fino a una concentrazione di 10^5parassiti·µl^(-1). Durante la tesi, è stato inoltre eseguito il primo esperimento di monitoraggio (follow up). Più tardi, un nuovo microchip con aree sensibili compenetrate è stato ideato, fabbricato e caratterizzato in modo da rimuovere il segnale spurio di drift tipico del chip precedente. Questa tesi è stata parte del progetto TMek. Le attività sperimentali sono state eseguite a PoliFAB, il centro di nano e micro tecnologia del Politecnico di Milano sotto la diretta supervisione dei Prof. Giorgio Ferrari e Prof. Riccardo Bertacco, direttore del gruppo "NABIS" del Dipartimento di Fisica del Politecnico di Milano, e all' Ospedale Luigi Sacco di Milano. Il sistema elettronico del dispositivo è stato sviluppato e caratterizzato in collaborazione con il gruppo "I3N" del Politecnico di Milano, diretto dai Prof. Marco Sampietro e Prof. Giorgio Ferrari. I campioni di sangue usati nei test di calibrazione sono stati forniti e trattati dal gruppo "µBS Lab" del Politecnico di Milano, guidato dal Prof. Gianfranco B. Fiore, mentre la campagna di validazione clinica è stata fatta nel laboratorio di diagnosi dell' Hôpital Saint Luc di Mbalmayo in Camerun.
Optimization of a lab-on-a-chip rapid diagnostic test for malaria
FAGIANI, FEDERICO
2018/2019
Abstract
Malaria remains the most important mosquito-borne infectious disease worldwide, with 3.5 billion people still at risk, 228 million cases and 405 000 deaths in 2018. The disease is caused by parasites belonging to the genus of Plasmodium, which infect red blood cells by feeding on haemoglobin and transforming it into hemozoin nanocrystals. Despite the World Health Organization recommends “prompt parasite-based diagnosis in all patients suspected of malaria before treatment is administrated, either by microscopy or malaria rapid diagnostic test (RDTs)”, the quality of microscopy-based diagnosis is frequently inadequate while the reliability of RTDs based on antigens detection is still affected by a sizable number of false negative/positive results. This master’s thesis project is about the optimization and characterization of a lab-on-a-chip pan-malaria rapid diagnostic test, that, based on the paramagnetic properties of hemozoin nanocrystals, combines magnetophoretic separation and electrical impedance detection. The presented diagnostic test, exploiting the competition between gravity and magnetic forces, relies on the local capture of infected red blood cells and hemozoin crystals on micromagnetic concentrators and subsequent detection by electrical measurement. The long-range attraction is provided by external NdFeB permanent magnets, while a concentration on the electrodes is performed by Ni pillars fabricated in the chip. Finally, because of the presence of particles within a solution changes its conductivity, the amount of hemozoin crystals and infected red blood cells can be quantified through an impedance variation detection. During this thesis work, different activities have been carried out to characterize and optimize a diagnostic platform previously developed. First, a solid and reliable analysis code has been realized and used during a preclinical validation study carried out in Cameroon. Then, other important characterization experiments have been performed to demonstrate a limit of detection (LoD) of 10parasites·µl^(-1) and a measurement range from the LoD up to 10^(5)parasites·µl^(-1). During the thesis, the first follow up monitoring has also been carried out. Later, a new microchip with interpenetrated sensitive regions has been designed, fabricated and characterized in order to remove the spurious drift signal affecting the previous chip. This thesis work was part of the TMek project. The experimental activities have been performed in PoliFAB, the micro and nanotechnology centre of “Politecnico di Milano”, under the direct supervision of Prof. Giorgio Ferrari and Prof. Riccardo Bertacco, head of the Nanomagnetism for Biology and Spintronics group of the Department of Physics, and in “Luigi Sacco” hospital in Milan. The detection system has been developed in collaboration with the "I3N" group of “Politecnico di Milano”, directed by Prof. Marco Sampietro and Prof. Giorgio Ferrari, that designed, fabricated and characterized the electronics of the device. The blood samples used in the calibration tests have been provided and treated by "µBS Lab" of "Politecnico di Milano”, led by Prof. Gianfranco B. Fiore, while the preclinical validation campaign has been carried out in the diagnostic laboratory of Hôpital Saint Luc of Mbalmayo in Cameroon.| File | Dimensione | Formato | |
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Tesi_Fagiani_finale.pdf
Open Access dal 07/04/2021
Descrizione: Testo della tesi finale
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https://hdl.handle.net/10589/165225