Development of Point-Of-Care Assay to Monitor Intestinal Bowel Disease Biomarkers

This thesis focuses on developing Point-Of-Care (POC) assays that could potentially be used to monitor the severity of Inflammatory Bowel Disease (IBD). We demonstrate our efforts to identify biomarkers that can characterize the severity of chronic inflammation in the gastrointestinal tract. The current clinical standard for monitoring IBD is endoscopic evaluations with biopsies. Patients do not like these frequent discomfort-inducing invasive procedures, underscoring the necessity for a non-invasive and cost-effective technique to monitor inflammation. In the first project, we focused on an enzyme biomarker, intestinal alkaline phosphatase (IAP), expressed in fecal samples of IBD patients. Stool samples containing IAP were mixed with the synthetic substrate, and IAP concentrations were detected using a personal glucose meter (GM). This rapid, non-invasive, inexpensive, user-friendly method provided the results within 15 min, with a limit of detection (LOD) of 0.0027 U. Importantly, this assay could differentiate between mice with active colitis and mice without colitis. The results were published in ACS Sensors. 2021, 6(3), 698-702. The second project focused on the Matrix Metalloproteinase-9 (MMP-9), an inflammatory biomarker implicated in IBD. The assay relies on an enzyme amplification strategy. In brief, MMP-9 reacted with its substrate and released thiol groups; these thiol groups activated inactivated papain, and the activated papain reacted with its substrate to generate a fluorescence signal. Unfortunately, a side reaction between the MMP-9 substrate and inactivated papain led to the discontinuation of the project. In the third project, we developed a signal enhancement method using biorthogonal chemistry and a layer-by-layer approach. Here, we focused on Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-10 (IL-10). Instead of using an enzyme and substrate as in commercial enzyme-linked immunoassays (ELISA), we used fluorescent dye-encapsulated silica nanoparticles as reporters and generated a layer-by-layer format using biorthogonal chemistry. We reached a LOD of TNF-ɑ of 73 fg/mL and IL-10 of 73 fg/mL, 137 fold and 120 fold higher in sensitivity, respectively, compared to ELISA results. In the fourth project, we conducted initial studies to evaluate if the assay could be translated to vertical flow assays. Our results indicate that this is possible; however, more research is needed