Development of a lab-on-a-chip microfluidic system for the detection of C-reactive protein in whole human blood and an antibody biomarker for the detection of Alzheimer's disease
This thesis is divided into two sections. The first section focuses on developing a better method for the detection of C - reactive protein (CRP) from whole blood. CRP plays a key role in determining the risks associated with various cardiovascular diseases. However, detecting this protein is a challenge since it accounts for < 1% of all known blood proteins. This protein can be detected by using a sandwich ELISA inside of a microfluidic chip made from Polydimethyl Siloxane (PDMS). PDMS chips are created that will first remove blood cells and platelets from whole blood. For the removal of blood cells, different filter papers that specialize in blood separation are tested based on the volume of filtrate recovered, amount of blood cells filtered out and whether the cells rupture or not. From these tests, it was determined that the Vivid Plasma Membranes GX and GR from the Pall Company was the most suitable filter based on blood cell removal and quality of filtrate. To further compliment this, these chips need to extract high abundance plasma proteins (HAP) including human IgG antibody and human serum albumin. These experiments focused on using protein A as a method for the removal of IgG due to its high affinity with the antibody. This will be then coupled with IgG that has been pre-attached to Protein A specifically modified to attach to Human Albumin. Experiments were conducted in order to obtain the appropriate incubation time needed for removal of antibody/protein as well as the effects on surface concentration and amount of incubation reservoirs. The final step was to combine these two aspects in order to create a microfluidic chip that accurately detects CRP by filtering out blood cells and removing high abundance proteins. This resulted in a microfluidic chip that was able to detect CRP from whole blood while filtering out blood cells and removing the bulk of the high abundant plasma proteins in order to obtain a high signal. This was performed by the addition of multiple incubation areas for high abundance protein removal embedded on the chip. Detection was carried out via fiber optic cables and a standard curve was created that associated a specific CRP blood concentration to an intensity found via fiber optics. By use of this method, a correlation between the concentration of CRP in a sample of blood, as well as a limit of detection for the system could be obtained. The second section focuses on identifying an antibody fragment as a bio-marker against tau protein. Tau protein is a known to play a role in the development of Alzheimer's disease. This is due to the rapid addition of multiple phosphate groups attaching to the protein. This section focuses on the identification of antibody fragments that detect a normal form of tau and one affected by the addition of phosphate groups. These fragments are recombinant antibody fragments created via phage display. Antibody phage is inserted into a well, in which the phage that is able to bind to tau will bind to it. These phages are then injected into an E. coli culture which will produce antibodies known as single-chain variable fragments (scFvs). These antibodies are extracted and tested on whether or not they attach to the specific tau antigens. After performing these experiments, two scFvs have been found that attach to both forms of tau: one with phosphate groups and one without. Titration curves and DNA sequences for both antibodies were also created. With these antibodies, they can be used as an effective biomarker into the early treatment of Alzheimer's disease.
Alex S Pytka,
"Development of a lab-on-a-chip microfluidic system for the detection of C-reactive protein in whole human blood and an antibody biomarker for the detection of Alzheimer's disease"
Dissertations and Master's Theses (Campus Access).