In this study, we applied protein probe technique which are molecules designed to bind to proteins, allowing researchers to study and manipulate proteins in biological systems. These probes are invaluable tools in molecular biology, biochemistry, and cell biology. Some common techniques involving protein probes include, Antibodies, Affinity Tags, Chemical Cross-Linking, Fluorescent Probes and more. In this study, our primary objective was to employ the highly sensitive and reliable TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) for the precise identification of individual cells within the sample. TR-FRET is an advanced method widely utilized in biomedical research and drug development, contributing significantly to High-Throughput Screening (HTS). Fluorescence Resonance Energy Transfer (FRET), is a crucial physical phenomenon increasingly harnessed in contemporary biomedical research and drug discovery. FRET operates on the principle of distance-dependent energy transfer from a donor molecule to an acceptor molecule, making it an invaluable tool for investigating molecular interactions. In this process, the donor molecule, typically a dye or chromophore, absorbs energy, which is subsequently transferred to the acceptor chromophore. The outcome of this energy transfer is a reduction in the donor's fluorescence intensity and excited state lifetime, coupled with an increase in the acceptor's emission intensity. Pairs of molecules exhibiting such FRET interactions are commonly referred to as donor/acceptor pairs. We integrated FRET into our study to gain detailed insights into molecular interactions, aiming to enhance our understanding of individual cells in the sampled population. This integration significantly boosts HTS, proving valuable in diverse areas of biomedical research and drug development due to its impressive sensitivity. The term "sensitivity" is crucial in analytical chemistry and diagnostic testing, indicating a method's capability to detect minute substance concentrations in a sample. Improving analytical sensitivity is key to advancing diagnostic technologies, especially in medical diagnostics, drug development, environmental monitoring, and forensic analysis. Enhanced analytical sensitivity is vital, enabling the detection of substances at lower concentrations and finding applications in early disease detection, environmental hazard monitoring, and industrial quality control. Our systematic approach involved optimizing conditions using a fluorescent-based method to heighten sensitivity, specifically to detect a lower number of cells, including individual cells in the sample. To achieve this, we labeled different peptides with a fluorescent dye (CY5) as a quencher and another probe as a chelate. The lanthanide properties of the chelate, such as a large Stoke shift, high quantum yield, and fluorescent stability, made them suitable for incorporation into the cell-based assay. In comparing FRET and QRET methods, strategically chosen for high sensitivity in Single Cell detection, we undertook labeling, purification, and confirmation of our probe. Exploration of assay conditions, coupled with reproducibility adjustments, led to the successful establishment of conditions enabling the detection of single cells. While numerous factors affect the FRET, hence our study focused on testing numerous condition to establishing these conditions to attain single-cell sensitivity. Subsequently, we applied and tested these conditions across various cell lines to demonstrate the method's reproducibility. This validation involved obtaining consistent results across different cell lines, confirming the robustness of our approach. To further bolster the credibility of our assay, we assessed linearity in diverse cell lines, consistently achieving positive outcomes.
In questo studio, abbiamo applicato la tecnica delle sonde proteiche, che sono molecole progettate per legarsi alle proteine, consentendo ai ricercatori di studiare e manipolare proteine nei sistemi biologici. Queste sonde sono strumenti inestimabili nella biologia molecolare, biochimica e biologia cellulare. Alcune tecniche comuni che coinvolgono le sonde proteiche includono anticorpi, etichette di affinità, legame chimico incrociato, sonde fluorescenti e altro ancora. In questo studio, il nostro obiettivo principale era impiegare la tecnica altamente sensibile e affidabile TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) per l'identificazione precisa delle singole cellule all'interno del campione. Il TR-FRET è un metodo avanzato ampiamente utilizzato nella ricerca biomedica e nello sviluppo di farmaci, contribuendo significativamente allo screening ad alto rendimento (HTS). Il Trasferimento di Energia per Risonanza di Fluorescenza (FRET) è un fenomeno fisico cruciale sempre più sfruttato nella ricerca biomedica contemporanea e nella scoperta di farmaci. Il FRET si basa sul principio del trasferimento di energia dipendente dalla distanza da una molecola donatrice a una molecola accettatrice, rendendolo uno strumento inestimabile per investigare le interazioni molecolari. Nel nostro studio, abbiamo integrato il FRET per ottenere dettagliate conoscenze sulle interazioni molecolari, mirando a migliorare la nostra comprensione delle singole cellule nella popolazione campionata. Questa integrazione potenzia significativamente lo screening ad alto rendimento, dimostrando di essere preziosa in diverse aree della ricerca biomedica e dello sviluppo di farmaci grazie alla sua notevole sensibilità. Il termine "sensibilità" è cruciale nella chimica analitica e nei test diagnostici, indicando la capacità di un metodo di rilevare concentrazioni minime di sostanze in un campione. Migliorare la sensibilità analitica è fondamentale per avanzare nelle tecnologie diagnostiche, specialmente nella diagnostica medica, nello sviluppo di farmaci, nel monitoraggio ambientale e nell'analisi forense. La nostra approccio sistematico ha coinvolto l'ottimizzazione delle condizioni utilizzando un metodo basato sulla fluorescenza per aumentare la sensibilità, in particolare per rilevare un minor numero di cellule, comprese le singole cellule nel campione. Per raggiungere questo obiettivo, abbiamo contrassegnato diversi peptidi con un colorante fluorescente (CY5) come estinguente e un'altra sonda come chelante. Le proprietà dei lantanidi del chelante, come uno spostamento di Stokes ampio, un alto rendimento quantico e una stabilità fluorescente, li hanno resi adatti all'incorporazione nel saggio basato sulle cellule. Nella comparazione tra i metodi FRET e QRET, scelti strategicamente per una elevata sensibilità nella rilevazione delle singole cellule, abbiamo proceduto all'etichettatura, purificazione e conferma della nostra sonda. L'esplorazione delle condizioni del saggio, insieme agli aggiustamenti di riproducibilità, ha portato alla riuscita stabilizzazione delle condizioni che consentono la rilevazione delle singole cellule. Sebbene numerosi fattori influenzino il FRET, il nostro studio si è concentrato sulla verifica di numerose condizioni per stabilire queste condizioni al fine di ottenere la sensibilità delle singole cellule. Successivamente, abbiamo applicato e testato queste condizioni su diverse linee cellulari per dimostrare la riproducibilità del metodo. Tale convalida ha comportato l'ottenimento di risultati coerenti su diverse linee cellulari, confermando la solidità del nostro approccio. Per rafforzare ulteriormente la credibilità del nostro saggio, abbiamo valutato la linearità in diverse linee cellulari, ottenendo in modo coerente risultati positivi.
Rilevamento di singole cellule mediante l'applicazione di tecniche basate sulla famiglia di sonde proteiche altamente sensibili alla fluorescenza - FRET e QRET
MASOUMI, AHMADREZA
2022/2023
Abstract
In this study, we applied protein probe technique which are molecules designed to bind to proteins, allowing researchers to study and manipulate proteins in biological systems. These probes are invaluable tools in molecular biology, biochemistry, and cell biology. Some common techniques involving protein probes include, Antibodies, Affinity Tags, Chemical Cross-Linking, Fluorescent Probes and more. In this study, our primary objective was to employ the highly sensitive and reliable TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) for the precise identification of individual cells within the sample. TR-FRET is an advanced method widely utilized in biomedical research and drug development, contributing significantly to High-Throughput Screening (HTS). Fluorescence Resonance Energy Transfer (FRET), is a crucial physical phenomenon increasingly harnessed in contemporary biomedical research and drug discovery. FRET operates on the principle of distance-dependent energy transfer from a donor molecule to an acceptor molecule, making it an invaluable tool for investigating molecular interactions. In this process, the donor molecule, typically a dye or chromophore, absorbs energy, which is subsequently transferred to the acceptor chromophore. The outcome of this energy transfer is a reduction in the donor's fluorescence intensity and excited state lifetime, coupled with an increase in the acceptor's emission intensity. Pairs of molecules exhibiting such FRET interactions are commonly referred to as donor/acceptor pairs. We integrated FRET into our study to gain detailed insights into molecular interactions, aiming to enhance our understanding of individual cells in the sampled population. This integration significantly boosts HTS, proving valuable in diverse areas of biomedical research and drug development due to its impressive sensitivity. The term "sensitivity" is crucial in analytical chemistry and diagnostic testing, indicating a method's capability to detect minute substance concentrations in a sample. Improving analytical sensitivity is key to advancing diagnostic technologies, especially in medical diagnostics, drug development, environmental monitoring, and forensic analysis. Enhanced analytical sensitivity is vital, enabling the detection of substances at lower concentrations and finding applications in early disease detection, environmental hazard monitoring, and industrial quality control. Our systematic approach involved optimizing conditions using a fluorescent-based method to heighten sensitivity, specifically to detect a lower number of cells, including individual cells in the sample. To achieve this, we labeled different peptides with a fluorescent dye (CY5) as a quencher and another probe as a chelate. The lanthanide properties of the chelate, such as a large Stoke shift, high quantum yield, and fluorescent stability, made them suitable for incorporation into the cell-based assay. In comparing FRET and QRET methods, strategically chosen for high sensitivity in Single Cell detection, we undertook labeling, purification, and confirmation of our probe. Exploration of assay conditions, coupled with reproducibility adjustments, led to the successful establishment of conditions enabling the detection of single cells. While numerous factors affect the FRET, hence our study focused on testing numerous condition to establishing these conditions to attain single-cell sensitivity. Subsequently, we applied and tested these conditions across various cell lines to demonstrate the method's reproducibility. This validation involved obtaining consistent results across different cell lines, confirming the robustness of our approach. To further bolster the credibility of our assay, we assessed linearity in diverse cell lines, consistently achieving positive outcomes.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
Per maggiori informazioni e per verifiche sull'eventuale disponibilità del file scrivere a: unitesi@unipv.it.
https://hdl.handle.net/20.500.14239/17323