Investigation of Gold Nanoprobe Extraction from Various Aqueous Two-Phase System Regions to Improve Lateral-Flow Immunoassay Detection of Protein and Bacterial Targets
The objective of this thesis was to investigate aqueous two-phase systems (ATPSs) as a concentration method for improving the lateral-flow immunoassay (LFA) detection of proteins and bacteria at the point-of-care (POC). In the first portion of the thesis, the polyethylene glycol (PEG)-potassium phosphate (salt) ATPS was used to concentrate transferrin (Tf), a model protein for C-reactive protein (CRP), to the interfacial region between the two bulk phases of the ATPS. In the second portion of the thesis, the PEG-salt ATPS was used to concentrate the bacterium Streptococcus mutans (S. mutans) into the bottom PEG-poor, salt-rich phase of the ATPS. For both studies, the ATPS concentration step was integrated with LFA in order to improve the detection of protein and bacterial targets. Our technologies therefore have the potential to improve POC solutions that require target concentration, such as detecting CRP and S. mutans in the respective oral disease diagnostic tests for periodontal disease and dental caries.
The detection of proteins at the POC allows for the chairside detection of diseases such as periodontal disease, which is associated with the presence of CRP in the body. A detection assay that is rapid, inexpensive, portable, and easy to use is ideal for this application, whereby the LFA is one such assay that fulfills these requirements. However, the sensitivity of LFA is inferior to laboratory-based assays, such as the enzyme-linked immunosorbent assay (ELISA), and needs to be improved. In order to improve the protein sensitivity of LFA, we utilized the PEG-salt ATPS to concentrate our model protein Tf prior to detection. Due to the size of proteins and other small biomolecules, these biomolecules will partition evenly between the two phases of the ATPS. To address this issue, we developed a novel approach that utilized larger colloidal gold nanoparticles decorated with anti-Tf antibodies that partitioned preferentially to the interfacial region between the two phases due to a delicate balance of the components comprising the nanoparticles. These nanoparticles bound to Tf and aided its transport to the interfacial region where Tf was consequently concentrated. Since the interfacial region represents a very small volume region that forms irrespective of the volume ratio, the volume ratio that reached equilibrium in the shortest time was chosen, reducing the sample extraction time to 10 min for phosphate-buffered saline (PBS) and to within 15-25 min for the complex solutions of fetal bovine serum (FBS) and synthetic urine. By concentrating Tf prior to LFA detection, the detection limit of LFA was improved by 100-fold from 1 ng/?L to 0.01 ng/?L in PBS, FBS, and synthetic urine. Thus, the ability to concentrate Tf bound to colloidal gold nanoparticles in a shorter duration of time provides a novel approach for improving LFA detection of small biomolecules.
POC detection of bacteria also allows for the chairside detection of diseases such as dental caries, which is caused by the presence of S. mutans. As in protein detection, the rapid time to result, low cost, portability, and ease of use make LFA an appropriate detection assay for the detection of bacteria. However, the bacterial sensitivity of LFA is inferior to laboratory-based methods, such as the use of cell culture with subsequent colony counts, polymerase chain reaction (PCR), and ELISA. Thus, we utilized the PEG-salt ATPS with an extreme volume ratio to concentrate S. mutans prior to LFA detection. Unlike proteins, S. mutans and other bacterial cells are larger in size and will experience greater steric, excluded-volume interactions with the PEG polymers present at higher concentrations in the PEG-rich, salt-poor phase of the ATPS, causing extreme partitioning to the PEG-poor, salt-rich phase. The S. mutans bacteria concentrated in the PEG-poor, salt-rich phase was extracted, mixed with colloidal gold nanoparticles decorated with anti-S. mutans antibodies, and applied to an LFA test strip. When S. mutans was concentrated prior to LFA detection, the detection limit of LFA was improved by slightly less than 10-fold from approximately 107 colony forming units (CFU)/mL to approximately 106 CFU/mL in PBS. The results of this study confirmed the applicability of using a PEG-salt ATPS to concentrate bacteria into one phase prior to extracting that phase for subsequent addition to an LFA test strip. Therefore, these results form the basis for future studies in which the longer phase separation time associated with extreme volume ratios will be addressed to allow for improved bacterial detection assay sensitivity within a shorter period of time.