In the context of biosensors, there is an increasing demand for multiple analyte (multiplex) devices that can quickly provide measurements for several analytes using a small-volume sample. In this experimental thesis, conducted as industrial collaboration with a local company, an in vitro assay was developed for measuring glucose, total cholesterol, and alanine aminotransferase (ALT), in view of integrating it with a portable reflectance spectrophotometer device. The design was based on a colorimetric assay using an enzymatic cascade while the final signal was measured using a spectrophotometer. Overall, three enzymatic cascades were produced, the first comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). GOx catalyzes the production of hydrogen peroxidase (H 2 O 2 ) in the presence of glucose, while HRP catalyzes the oxidation of the substrates DAOS (sodium 3-((3,5-dimethoxyphenyl)(ethyl)amino)- 2-hydroxypropane-1-sulfonate) and 4-APP (4-amine antipyrine) in the presence of H 2 O 2 , generating a quinoneimine dye and a change of color. Total cholesterol required an additional step, since its presence in biological fluids (e.g., blood) is often associated with esterification with fatty acids. Therefore, cholesterol esterase (CE) was used in the first step, producing free cholesterol molecules. The second and third steps involved the use of cholesterol oxidase (COx) and HRP, reproducing similar catalytic steps, as for glucose. Finally, ALT was measured using a different approach. In the presence of a-ketogluarate and L-alanine, ALT catalyzed the formation of pyruvate that was then oxidized by pyruvate oxidase (POx) producing H 2 O 2 . In the final step, similarly to the other two analytes, HRP was used to catalyze the oxidation of the final substrate DAOS, resulting in a color change. All systems were optimized in vitro, and a final test was performed in simulated blood serum. Results showed good linearity of the systems and limits of detection well within physiological glucose, ALT, and total cholesterol values. These assays could be integrated in a portable device by immobilizing the enzymes on a solid support (e.g., cellulose) and using capillary forces and a single wavelength reflectance spectrophotometer to realize a multiplex biosensor.
Nel contesto dei biosensori vi è una crescente domanda di dispositivi multi-analita (multiplex) in grado di fornire rapidamente misurazioni per diversi analiti utilizzando un campione di piccolo volume. In questa tesi sperimentale, il cui lavoro sperimentale è stato condotto come collaborazione aziendale con una impresa locale, è stato sviluppato un test in vitro per la misurazione di glucosio, colesterolo totale e alanina aminotransferasi (ALT), in vista della sua integrazione con uno spettrofotometro portatile a riflettanza. Il progetto si è basato un test colorimetrico che utilizza una cascata enzimatica, mentre il segnale finale è stato misurato utilizzando uno spettrofotometro. Complessivamente, sono state prodotte tre cascate enzimatiche, la prima comprendente glucosio ossidasi (GOx) e perossidasi di rafano (HRP). GOx catalizza la produzione di perossido di idrogeno (H₂O₂) in presenza di glucosio, mentre HRP catalizza l'ossidazione di un substrato finale DAOS (sodio 3-((3,5-dimetssifenil)(etil)ammino)-2- idrossipropano-1-sulfonato) e in presenza di H₂O₂, con conseguente variazione di colore. Il colesterolo totale ha richiesto un passaggio aggiuntivo, poiché la sua presenza nei fluidi biologici (ad esempio, il sangue) è spesso associata all'esterificazione con acidi grassi. Pertanto, nel primo passaggio è stata utilizzata colesterolo esterasi (CE), producendo molecole di colesterolo libero. Il secondo e il terzo passaggio hanno comportato l'uso di colesterolo ossidasi (COx) e dell'HRP, riproducendo passaggi catalitici simili a quelli per l’analisi del glucosio. Infine, l'ALT è stato misurato utilizzando un approccio diverso. In presenza di α-chetogluarato e L- alanina, ALT catalizza la formazione di piruvato, che è stato poi ossidato da piruvato ossidasi (POx), producendo H₂O₂. Nel passaggio finale, analogamente agli altri due analiti, HRP catalizza l'ossidazione del substrato finale DAOS, con conseguente cambiamento di colore. Tutti i sistemi sono stati ottimizzati in vitro ed è stato eseguito un test finale in siero sanguigno simulato. I risultati hanno mostrato una buona linearità dei sistemi e limiti di rilevazione ampiamente entro i valori fisiologici di glucosio, ALT e colesterolo totale. Questi test possono essere integrati in un dispositivo immobilizzando gli enzimi su un supporto solido (ad esempio, cellulosa) e utilizzando forze capillari e uno spettrofotometro a riflettanza a lunghezza d'onda singola per realizzare un biosensore multiplex, portatile ed economico.
A Multiplex Biosensor for Glucose,Cholesterol, and Alanine Aminotransferase: A Preliminary Study
TIMAR, HAMED
2024/2025
Abstract
In the context of biosensors, there is an increasing demand for multiple analyte (multiplex) devices that can quickly provide measurements for several analytes using a small-volume sample. In this experimental thesis, conducted as industrial collaboration with a local company, an in vitro assay was developed for measuring glucose, total cholesterol, and alanine aminotransferase (ALT), in view of integrating it with a portable reflectance spectrophotometer device. The design was based on a colorimetric assay using an enzymatic cascade while the final signal was measured using a spectrophotometer. Overall, three enzymatic cascades were produced, the first comprising glucose oxidase (GOx) and horseradish peroxidase (HRP). GOx catalyzes the production of hydrogen peroxidase (H 2 O 2 ) in the presence of glucose, while HRP catalyzes the oxidation of the substrates DAOS (sodium 3-((3,5-dimethoxyphenyl)(ethyl)amino)- 2-hydroxypropane-1-sulfonate) and 4-APP (4-amine antipyrine) in the presence of H 2 O 2 , generating a quinoneimine dye and a change of color. Total cholesterol required an additional step, since its presence in biological fluids (e.g., blood) is often associated with esterification with fatty acids. Therefore, cholesterol esterase (CE) was used in the first step, producing free cholesterol molecules. The second and third steps involved the use of cholesterol oxidase (COx) and HRP, reproducing similar catalytic steps, as for glucose. Finally, ALT was measured using a different approach. In the presence of a-ketogluarate and L-alanine, ALT catalyzed the formation of pyruvate that was then oxidized by pyruvate oxidase (POx) producing H 2 O 2 . In the final step, similarly to the other two analytes, HRP was used to catalyze the oxidation of the final substrate DAOS, resulting in a color change. All systems were optimized in vitro, and a final test was performed in simulated blood serum. Results showed good linearity of the systems and limits of detection well within physiological glucose, ALT, and total cholesterol values. These assays could be integrated in a portable device by immobilizing the enzymes on a solid support (e.g., cellulose) and using capillary forces and a single wavelength reflectance spectrophotometer to realize a multiplex biosensor.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14247/26315