Design and development of a site specific protein patterning technique for use in a microfluidic antibody separation device

Abstract

The rapid quantification of the concentration of different immunoglobulins classes from patient serum is required to diagnose patients in the early stages of sepsis. Microfluidic point of care technology can improve diagnostics by decreasing the analysis time, and integrating parallel analysis in a single portable device. The design of a novel method to fabricate surfaces presenting multiple micron scale protein motifs, for integration within a microfluidic channel device, is described in this thesis.

Initial research focussed on conjugating protein motifs on silicon <100> substrates in micron and submicron scale patterns. A method involving the UV-initiated conjugation of a heterobifunctional linker, undecylenic acid N-Hydroxysuccinimde ester (UANHS), to a hydrogen terminated silicon surface was investigated. A photolithographic mask and phase mask were used to form micron and submicron UANHS motifs respectively, on silicon. The conjugation of protein with UANHS motifs was investigated to determine how reproducible the patterns were. The conjugation of streptavidin, streptavidin-FITC, NeutrAvidin, single domain protein L and multidomain protein L to silicon surfaces, upon reaction with UANHS, were investigated. Fluorescently labelled probes that associated with the protein motifs were used to confirm successful conjugation of protein to the silicon. Micron scale motifs of streptavidin, streptavidin-FITC, NeutrAvidin and single domain protein L could be formed reproducibly on silicon. Using a phase mask 140 nm motifs of streptavidin-FITC, conjugated to silicon, were achieved.

Also an alternative method to pattern multiple proteins onto glass surfaces was investigated. A 500,000 MW dextran was modified to incorporate an aryl azide moiety, which was subsequently immobilised on glass surfaces. A method to synthesise and characterise the aryl azide conjugated dextran was investigated, as well as methods to characterise and improve the reproducibility of the aryl azide conjugated dextran layer immobilised on the glass surface. Two photolithographic masks and glass surfaces with alignment marks were fabricated. The masks were used to form micron scale protein motifs, via a photoinitiated conjugation reaction, on the aryl azide conjugated dextran surface. An in-house alignment system was built and a method to produce adjacent protein motifs was investigated. Two adjacent micron scale patterns of multidomain protein L and protein A were achieved. The surface density of conjugated protein L was investigated and a density of ~1.16x10¹¹ molecules/cm² was confirmed. This approach offers a method to attach high density micron scale protein motifs, aligned with micron scale resolution, which is vital to the realisation of a microfluidic point of care device.

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