OPTIMIZATION OF LIPOSOME-ENHANCED HIGH THROUGHPUT BIOASSAYS
High throughput analysis is important for the rapid screening or quantitative analysis of large number of samples Microtiter plates provide an excellent platform for high throughput assays and have been used for decades in immunological and nucleic acid assays. Work focused here on the adaptation of nucleic acid bioassays with liposome amplification in a microtiter plate format with two main objectives: (1) optimization of protein-liposome coupling chemistry and (2) optimization of DNA immobilization in microtiter plates. The protein streptavidin was coupled to COOH-labeled liposomes using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide*HCl (EDC) chemistry. This procedure was optimized with respect to liposome encapsulation efficiency (total lipid concentration over encapsulated dye concentration) and binding efficiency. For the optimization of DNA probe immobilization in microtiter plates, five different plate types were investigated; medium binding, high binding, NeutrAvidin coated, and two different types of amine binding. Assay protocols for each plate were developed in addition to the ability to immobilize DNA probes and the effect on both the target sequence hybridization and liposome binding. In all cases, data was recorded using a fluorescence plate reader or dynamic light scattering. Optimal conditions for the coupling of streptavidin to COOH-labeled liposomes were determined as 66% liposomes in MES buffer at pH 7 and a streptavidin concentration of 0.05 mol% of the liposome phospholipid concentration. A coupling time of 15 minutes was sufficient. In the case of the DNA probe immobilization in microtiter plates, a probe concentration of 1 to 50 nM enabled target sequence hybridization in all plates, with a maximum signal to noise ratio (S:N) at about 50 nM. In the case of DNA-labeled liposomes only the NeutrAvidin plates generated a signal, with a maximum S:N of about 400:1. In contrast, for streptavidin-labeled liposomes, the NeutrAvidin plates generated no signal, but the high-binding adsorption plates generated a maximum S:N of about 80:1 (in all cases at 50 nM of target sequence). The covalent binding plates generated a S:N of only 5:1 to 10:1. Following from these results, it is recommended that the NeutrAvidin plate be used with DNA-labeled liposomes to perform a specific test and that the high binding adsorption plate be used with streptavidin-labeled liposomes to achieve a universal assay format.
Liposome; Microtiter Plate; DNA Immobilization; Streptavidin
dissertation or thesis