Development of Immunoassay Using Graphene and Microfluidic Platforms.

Abstract

Protein, as one of the most important functional biomolecules in the human body, plays a significant role in physiological responses and molecular diagnostics. Detecting the existence of proteins, quantifying concentration, and identifying protein types are therefore important techniques in many fields. Immunoassays are one of the major techniques relied on for protein detection. Immunoassays have been broadly applied in disease diagnosis, pharmaceutical development, food science, and environmental protection. The first part of this dissertation describes studies aimed at developing chemical vapor deposition (CVD) graphene as a large size protein biosensing platform. To utilize graphene as a biosensing platform, techniques to immobilize proteins on graphene are critical. In this dissertation work, carboxyl functional groups (-COOH) were created by graphene functionalization, and the functionalized graphene was characterized using Raman spectroscopy, X-ray photo spectroscopy (XPS), and fluorescence microscopy. The approach developed here provides information about protein coupling density and uniformity on large scale graphene (> cm2). The second and the third parts of the thesis describe the application of a microfluidic technique to two widely used protein detection methods – immunoblotting and dot blotting. The microfluidic systems were designed and fabricated to be easily interfaced with a common type of protein blotting membrane called polyvinylidene fluoride (PVDF) membrane. The microfluidic device was specifically applied to the antibody incubation step, which reduces antibody consumption and therefore also significantly reduces the cost of the assay. In microfluidic immunoblotting, an approach to activate the PVDF membrane to increase its protein binding capacity was developed. This was achieved by adding a surfactant Tween-20 to the antibody solution. The concentration of Tween-20 was optimized so that only the portion of the membrane within the channel region was activated. The system has been shown to be able to profile inflammatory signaling pathways. In microfluidic dot blotting, the influence of substrate hydrophobicity and protein concentrations on device design constraints were studied. Inflammatory cytokine detection using the developed microfluidic dot blotting system was determined. Altogether these experiments demonstrate that applying microfluidic techniques to protein immunoblotting and dot blotting improves detection efficiency, and reduces cost by utilizing less antibodies.