Miniaturization of heterogeneous immunoassays for the detection of microbial pathogens.
Published: 2005
Language: eng
Number of pages: 162
Description: Escherichia coli O157:H7 and Helicobacter pylori  are human gastrointestinal pathogens which can result in serious diseases. Infected patients develop strong immune responses which provide the basis for immune diagnostics. Currently, enzyme-linked immunosorbent assay (ELISA) is bench-top based. Miniaturization would prove beneficial because of the potential for automation, multiplexing, portability, and being less costly. The objectives of this thesis were to: (1) develop a hand-held ELISA to detect E. coli O157:H7 and (2) develop poly(dimethylsiloxane) (PDMS) based microfluidic immunoassays to detect both H. pylori and E. coli O157:H7 without using sophisticated equipment. Pressure-driven flow was used for solution delivery.In the second part, a miniaturized ELISA was developed by using PDMS microchannels. It reliably detected H. pylori antigens on the order of 10 ng. Analysis of 20 human serum samples of known H. pylori infection status demonstrated that this assay was highly sensitive and specific with an overall accuracy of 95%. A software package was developed to accurately quantify colour changes in microchannels while minimizing human subjectivity.In the first part of this thesis, a modular hand-held ELISA prototype was developed. Commercially available primary and secondary antibodies and a precipitating colour substrate were used. Results obtained using this prototype were comparable to conventional ELISA, which has a detection limit of 4 ng of E. coli O157:H7 antigens by using the naked eye. Furthermore, the PDMS solid substrate can be re-used, without losing sensitivity.In the third part, a novel label, resonant light-scattering gold nanoparticles of 80 nm in diameter, was used as a read-out for the microchannel immunoassay. Using dark-field microscopy, this assay detected H. pylori and E. coli O157:H7 antigens on the order of 10 ng. In addition, the nanoparticles within the microchannels can be stored for at least eight months without lost of signal intensity, thereby providing a means for long-term preservation.In summary, miniaturized immunoassays were developed using both hand-held and micro fluidic-based formats. The studies also show, for the first time, that PDMS can be used as a solid substrate for microbiological and human samples. They now can be further miniaturized by using photolithography and solution delivery can be automated by using electroosmotic flow.
Notes: Thesis (Ph.D.)--University of Toronto, 2005.

Electronic version licensed for access by U. of T. users.

Source: Dissertation Abstracts International, Volume: 66-06, Section: B, page: 2944.