Improving the Lateral-Flow Immunoassay Using Aqueous Two-Phase Systems

The objective of this thesis was to investigate a concentration method using aqueous two-phase systems (ATPS) for improving the detection of proteins and viruses at the point-of-care. In the first part of the thesis, aqueous two-phase micellar systems were generated using Triton X-114 surfactant to concentrate a model protein, namely transferrin (Tf). In the second part of the thesis, aqueous two-phase polymer-salt systems were generated using polyethylene glycol (PEG) and potassium phosphate salt to concentrate a model virus, namely bacteriophage M13 (M13). In both studies, the concentration step was combined with a detection assay, namely the lateral-flow immunoassay (LFA), to enhance the detection of protein and viral targets.The detection of proteins at the point-of-need has several applications such as detecting food allergens in a food sample and protein toxins used as biowarfare agents in-field. For such applications, a sensitive, yet rapid, inexpensive, and portable detection assay that requires minimal training and power is desired. Due to its ease of use, rapid processing, and minimal power and laboratory equipment requirements, the LFA is an appropriate assay for such applications. However, the LFA detection limit for proteins is inferior to lab-based assays, such as the enzyme-linked immunosorbent assay (ELISA), and needs to be improved. Aiming to improve the protein sensitivity of the LFA, we employed an aqueous two-phase micellar system composed of Triton X-114 surfactant to concentrate Tf prior to the detection step. However, one challenge with concentrating small biomolecules, such as proteins, is that they partition evenly between the two phases due to experiencing fewer excluded-volume interactions compared to larger biomolecules. To address this issue, we developed a novel approach involving larger colloidal gold nanoparticles decorated with anti-Tf antibodies in the concentration step to bind Tf and aid its transport to the micelle-poor phase. By manipulating the volume ratio of the two coexisting micellar phases to achieve higher concentrations, the Tf detection limit of LFA was improved by 10-fold from 0.5 i? ½g/mL to 0.05 i? ½g/mL. The ability to concentrate colloidal gold nanoparticles bound to Tf has opened up a whole new approach for improving the detection of smaller analytes with the LFA.Viral detection in the point-of-care setting also has several applications. For example, the detection of infectious viral agents and pandemic pathogens, such as the swine-origin influenza A (H1N1) virus, is crucial for isolating confirmed cases and preventing outbreaks. The portability, simple operation procedure, rapid time to result, and minimal power and laboratory equipment requirements of the LFA make it an appropriate detection assay for such diagnostic applications. However, the viral sensitivity of the LFA is inferior to laboratory-based methods, such as viral culture and reverse-transcriptase polymerase chain reaction (PCR). We previously showed in a proof-of-principle study that the viral detection limit of the LFA could be improved by concentrating a model virus, namely bacteriophage M13, using an aqueous two-phase micellar system prior to the detection step. The previous investigation represented the first time these two established technologies were ever combined. However, the micellar system exhibited slow phase separation times that were on the order of hours, indicating a need to improve the speed of the concentration step. Therefore, in this study, we investigated an aqueous two-phase polymer-salt system composed of polyethylene glycol (PEG) and potassium phosphate salt, which phase separates on the order of minutes, to concentrate M13. Furthermore, the colloidal gold nanoparticles used as the colorimetric indicator in the LFA were modified with a coated layer of PEG in order to improve their stability in the high salt content of the PEG-salt system. When M13 was concentrated using the PEG-salt system and combined with the LFA, the detection limit was improved by 10-fold from 5x108 plaque forming units (pfu)/mL to 5x107 pfu/mL. This study represents the first time that viral detection by LFA has been combined with a concentration method using an aqueous two-phase polymer-salt system. The faster phase separating ability of the PEG-salt system is a significant advance for applying this concentration method to improving point-of-care detection. Furthermore, the viable function of the modified colloidal gold nanoparticles coated with PEG in the LFA demonstrates a novel method for detecting biomolecules in ATPS containing high levels of salt.