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Polymeric Microfluidic Plattform for Immunodetection
Auteur(s)
Linder, Vincent
Editeur(s)
De Rooij, Nicolas F
Résumé
The overall goal of this work was to develop a single-use microfluidic device for the immunological detection of clinically relevant species, such as human IgG. Poly(dimethylsiloxane) (PDMS) was chosen as a material for channel fabrication because of its optical transparency, ease of use and possible modification with a stable physisorption-based coating. Electrokinetically driven sample transport was used for its ease of tuning individual fluid flow velocities in the channel network. Moreover, electrokinetic effects are responsible for transporting analyte and tracer at different velocities. For the purpose of developing an electrokinetically driven assay in a PDMS/glass microfluidic platform, electroosmosis in microchannels was studied. Several physico-chemical treatments of the PDMS surface prior to sealing on glass were considered. The hydrophobic PDMS surface was shown to suffer from severe adsorption, and therefore the deposition of a passivating surface coating was investigated. Integration of biotinylated probes was possible in the outer layer of the three-layer biotin/neutravidin-based sandwich coating. Selective deposition of biotinylated anti-human IgG in the microfluidic platform was developed using a laminar flow-based patterning technique. The feasibility of a heterogeneous immunoreaction between anti-human IgG immobilized in the channels and Cy5-labeled human IgG in an electrokinetically transported liquid sample was demonstrated. Qualitative monitoring of the bound Cy5-human IgG was achieved by fluorescence microscopy. Quantitative measurements with a fluorescence scanner originally designed for arrays required changes in the chip design. Reproducibility and confidence in the assay results were enhanced by the implementation of an internal standard. This was done by co-immobilizing biotinylated anti-mouse IgG along with anti-human IgG in the biospecific region of the chip. A competitive immunoassay for human IgG was set up, using Cy5-human IgG as bound tracer and Cy3-mouse IgG as internal standard. The ratio of Cy5 to Cy3 signal was used to assess the original human IgG concentration, while absence of Cy3 signal was indicative of faulty chip function. Human IgG concentration dependence was investigated under physiological and alkaline conditions. Finally, monitoring of serum human IgG levels of patients and its potential application to diagnostics was investigated
Notes
Thèse de doctorat : Université de Neuchâtel : 2001 ; 1602
Type de publication
Resource Types::text::thesis::doctoral thesis
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