Publicação
Development of a phage-based lab-on-chip for the multiplex detection of bloodstream pathogens
| Resumo: | Bloodstream infections (BSIs) are characterized by high incidence and mortality and thus can greatly impact healthcare systems. The prompt administration of antibiotics along with their de-escalation as early as possible are key elements for BSIs effective control. One of the main constraints that limit these actions is the difficulty in detecting the BSIs’ causative agents. The current diagnostic approaches suffer from low specificity, high costs, lack of portability, are time-consuming, and/or require trained personnel for their execution. Thus, it is of paramount importance the development of novel robust methods. Hence, to address this concern, the main goal of this work was to develop a bacteriophage-based lab-on-chip (LoC) platform for multiplex detection of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, which are highly prevalent isolates from BSIs cases. In order to achieve the goal, phage-based probes that present outstanding high specificity, sensitivity, and low-cost production were designed and fused with fluorescent proteins to be used as bacteria recognition elements. These were combined with the advantageous properties of microfluidic devices, such as miniaturization, low sample volume, and portability along with optical sensors due to their sensitivity and easy integration in LoC devices, allowing bacterial detection. In this scope, firstly, a reporter phage with a green fluorescent protein (GFP) for E. coli detection was successfully constructed, nonetheless, it showed a weak fluorescence signal, making unfeasible its use in further experiments. As a surrogate strategy, phage proteins, namely receptor-binding proteins (RBPs) and cell-wall binding domains (CBDs), were developed and fused with fluorescence proteins. Their functional analysis (binding efficiency, sensitivity, and specificity in whole blood) was evaluated using different techniques such as flow cytometry and spectrofluorimetry. In this sense, three recombinant proteins were built: a CBD fused with a GFP that showed to be efficient in the detection of 1-5 CFU/mL of S. aureus after blood culture; an E. coli RBP fused with mCherry that recognized cells in distinct viability states and detected bacteria in various human biological specimens; an RBP for P. aeruginosa fused with mAmetrine that revealed to be very specific. The RBPs for P. aeruginosa and E. coli were used as probes in a bead-based microfluidic assay for the multiplex detection of both pathogens in whole blood. This miniaturized assay detected 104 CFU/mL in 70 min without enrichment, showing bacterial quantitative capabilities. Lastly, an optical setup based on photodetectors was developed and combined with the phage-based microfluidic assay, allowing the detection of P. aeruginosa in whole blood. Noteworthy, this system presented high specificity, and fastness, and is amenable to being fully automated to be used as a point-of-care device for BSIs diagnosis in healthcare settings. |
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| Autores principais: | Costa, Susana Patrícia Chaves |
| Assunto: | Bloodstream infections Microfluidics Phages Phage proteins Photodiodes Bacteriófagos Fotodíodos Infeções no sangue Microfluídica Proteínas fágicas |
| Ano: | 2023 |
| País: | Portugal |
| Tipo de documento: | tese de doutoramento |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | Bloodstream infections (BSIs) are characterized by high incidence and mortality and thus can greatly impact healthcare systems. The prompt administration of antibiotics along with their de-escalation as early as possible are key elements for BSIs effective control. One of the main constraints that limit these actions is the difficulty in detecting the BSIs’ causative agents. The current diagnostic approaches suffer from low specificity, high costs, lack of portability, are time-consuming, and/or require trained personnel for their execution. Thus, it is of paramount importance the development of novel robust methods. Hence, to address this concern, the main goal of this work was to develop a bacteriophage-based lab-on-chip (LoC) platform for multiplex detection of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus, which are highly prevalent isolates from BSIs cases. In order to achieve the goal, phage-based probes that present outstanding high specificity, sensitivity, and low-cost production were designed and fused with fluorescent proteins to be used as bacteria recognition elements. These were combined with the advantageous properties of microfluidic devices, such as miniaturization, low sample volume, and portability along with optical sensors due to their sensitivity and easy integration in LoC devices, allowing bacterial detection. In this scope, firstly, a reporter phage with a green fluorescent protein (GFP) for E. coli detection was successfully constructed, nonetheless, it showed a weak fluorescence signal, making unfeasible its use in further experiments. As a surrogate strategy, phage proteins, namely receptor-binding proteins (RBPs) and cell-wall binding domains (CBDs), were developed and fused with fluorescence proteins. Their functional analysis (binding efficiency, sensitivity, and specificity in whole blood) was evaluated using different techniques such as flow cytometry and spectrofluorimetry. In this sense, three recombinant proteins were built: a CBD fused with a GFP that showed to be efficient in the detection of 1-5 CFU/mL of S. aureus after blood culture; an E. coli RBP fused with mCherry that recognized cells in distinct viability states and detected bacteria in various human biological specimens; an RBP for P. aeruginosa fused with mAmetrine that revealed to be very specific. The RBPs for P. aeruginosa and E. coli were used as probes in a bead-based microfluidic assay for the multiplex detection of both pathogens in whole blood. This miniaturized assay detected 104 CFU/mL in 70 min without enrichment, showing bacterial quantitative capabilities. Lastly, an optical setup based on photodetectors was developed and combined with the phage-based microfluidic assay, allowing the detection of P. aeruginosa in whole blood. Noteworthy, this system presented high specificity, and fastness, and is amenable to being fully automated to be used as a point-of-care device for BSIs diagnosis in healthcare settings. |
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