Immunoassay, as a technique that utilizes antibody-antigen interactions for the detection of relevant analytes, plays an important role in our daily life. It can be used for the quantification of proteins and small molecules in a number of different fields, such as medical diagnosis, food safety and environmental monitoring. The analysis of antibodies directed against specific epitopes with high specificity and sensitivity in human serum is becoming an increasingly critical task in medical and life sciences as the process provides detection of a serologic biomarker which enables early diagnosis in many diseases: for example, infectious diseases such as HIV and hepatitis B, autoimmune diseases such as systemic lupus erythematosus (SLE), as well as allergies and cancers. Recently, there i...[ Read more ]

Immunoassay, as a technique that utilizes antibody-antigen interactions for the detection of relevant analytes, plays an important role in our daily life. It can be used for the quantification of proteins and small molecules in a number of different fields, such as medical diagnosis, food safety and environmental monitoring. The analysis of antibodies directed against specific epitopes with high specificity and sensitivity in human serum is becoming an increasingly critical task in medical and life sciences as the process provides detection of a serologic biomarker which enables early diagnosis in many diseases: for example, infectious diseases such as HIV and hepatitis B, autoimmune diseases such as systemic lupus erythematosus (SLE), as well as allergies and cancers. Recently, there is an urgent global need for point-of-care immunoassays which can be operated by end users since early stage antibody detection plays a crucial role in disease diagnosis and treatment.

In this thesis work, we developed a new on-chip immunoassay platform, which combines the yeast surface display (YSD) technique and a microfluidic system. By directly counting genetically-engineered yeast cells bound with target analytes, the immunoassay was realized on a microchip tailored for point-of-care applications. Taking advantage of YSD, antigenic reporters are easily to be obtained by simply yeast culture, and the tedious steps of antigen purification and wet-chemistry processes to immobilize probe antigen can be eliminated. At the same time, with individual cells readily counted by routine microscopy, fewer numbers of cells can be detected than fluorescence or chromophore-based methods. Thus, this method has the inherent characteristic to be highly sensitive. Furthermore, controlled laminar flow can be applied to preferentially remove nonspecifically bound cells, improving the selectivity of the assay.

We have built a prototypical microfluidic immunoassay device incorporating functionalized planar surface and fluidic force discrimination, and demonstrated multiplexed antibody detection using bifunctional engineered yeast cells. The magnetic bead-assisted method, which utilizes protein G coupled magnetic beads to capture antibody-labeled cells, improves the sensitivity of the YSD-based microfluidic immunoassay by 100 times and provides a detection limit of 0.5 ng/ml. Alternatively, we have also developed a functionalized micro pillar array (MPA)-based microfluidic immunoassay, utilizing capillary force as the driven power to replace pumping. The high surface area of the MPA results in a high possibility for cells to contact and attach to the pillars, and provides a detection limit of 5 ng/ml. This method is believed to have excellent compatibility with common biopsy used in general hospitals, as well as high potential to be integrated into a portable system.

In order to achieve real point-of-care antibody detection that can be operated by end users, we have developed a non-optical YSD based microfluidic immunoassay using micro Coulter particle counter (μCPC) to count yeast cells bound with target analytes. Preliminary study has demonstrated that oil droplets, latex particles and yeast cells can be counted by our approach.

As a synergistic combination of yeast surface display and a microfluidic system, this YSD based immunoassay has a high potential to be developed into a robust antibody detection tool, which can be integrated into microfluidic detection systems to enable portable analysis in the future.