Detection of Lysophosphatidic Acid in Serum; Towards a Cost Effective Test for Ovarian Cancer

Ovarian cancer is a disease that affects a quarter of a million new women and causes over 140,000 deaths worldwide annually. This is primarily due to how difficult the disease is to detect, with few symptoms present in the early stages of the disease and difficult to physically feel masses. Currently detection of ovarian cancer requires time consuming and expensive imaging studies such as transvaginal ultrasound and MRI scans, which are only performed if it is already suspected that a woman has the disease. The only currently accepted blood test for ovarian cancer detects the biomarker CA-125, but this test is only sensitive to 50% of cases and as a result is rarely used for signaling the presence of the cancer. There is another biomarker for ovarian cancer called lysophosphatidic acid (LPA) which has a sensitivity and specificity of over 90% for the disease, making it promising for use in testing for ovarian cancer. There are currently no low cost or easy to perform blood tests for LPA, preventing its use as a general test that can be performed at yearly physicals. However there is a duel protein system comprised of Gelsolin and Actin which is interrupted by interactions with LPA, which can therefore be used to develop a low cost blood test for the marker. These proteins could be bound to a biosensing surface, and the release of actin as a result of LPA would generate a measurable signal. Studying the fouling behaviour of LPA and serum on transverse shear mode biosensors, also known as quartz crystal microbalances, showed that the background fouling signal was too large for a test to be developed on this class of devices. Instead a test based on colorometric sensing of dye modified actin was conceived and evaluated. This test comprised of the protein gelsolin being bound to solid phase in a way capable of holding onto the protein in serum samples. The gelsolin was also be bound to a fluorescent dye modified actin that could be measured using colorimetric absorbance or fluorescence. These proteins were successfully created, and shown to create a complex that could be broken by LPA in a concentration dependent manner. Immobilization of this protein complex was performed on silica gel, using Ni-NTA which was attached to the surface via a trichlorosilane linker functionalized with an acid chloride head group, which was first synthesized in these experiments. Several conditions in the development of the surface Ni-NTA adlayer and subsequent protein immobilization were evaluated, and it was found that such things as a large pore size in the silica gel, removal of excess toluene from the surface following adlayer addition, and adequate washing and drying of the silica were all of crucial importance to the performance of this test in serum. Development of this test showed promise in quantifying LPA at biologically relevant concentrations, however more work needs to be done to lower the limit of detection to a point below the cut-off level for ovarian cancer. Crucially this test is very cheap to produce, and requires little human labour to perform which are characteristics required for its wide scale adoption in ovarian cancer screening.Ph.D