A quantum dot-based diagnostic immunoassay for biomarker detection in gastrointestinal inflammatory diseases

David Richard Hansberry

doi:10.17918/etd-3640

Advances and integration in the fields of molecular biology, nanotechnology, bioanalytical chemistry, nanofluidics, and electrochemistry have paved the way for more sensitive, specific and robust biosensors with wide ranging applications from bioterrorism to clinical medicine. In this thesis, we optimize a novel, in-house developed poly(methyl methacrylate), microcapillary immunoassay known as QLISA, in an attempt to quantify fecal levels of myeloperoxidase and lactoferrin to aid in differentiating inflammatory bowel disease (ulcerative colitis and Crohn's disease) from irritable bowel syndrome. Noninvasive specimen collection and analysis to help physicians distinguish these two similarly presenting diseases would allow more rapid and appropriate treatment. Given the similar clinical presentations and drastically different treatments, there is the potential to improve quality patient care while reducing both direct and indirect associated cost through the elimination of unnecessary procedures and more efficient medical treatment. Using a crosslinking strategy of branched, di-amino polyethylene glycol polymer and glutaraldehyde following wet chemical treatment with sodium hydroxide, we demonstrated significant increase in primary antibody capture on a PMMA substrate, as used in QLISA, over traditional passive adsorption methods typically used in commercial ELISA kits. This unique crosslinking approach provides potential for improved immunoassay performance and is not limited to QLISA or even PMMA-based biosensors. QLISA was validated against commercial ELISA kits when testing for myeloperoxidase and lactoferrin in human fecal samples of patients with an inflammatory bowel disease or healthy control. We found that myeloperoxidase levels were elevated from about 80 to over 12,000 times that of our healthy control patients. Similarly we found lactoferrin levels were elevated from about 265 to almost 9,000 times that of the control patients. Further analysis shows that levels of greater than 1ug/g of myeloperoxidase suggested inflammatory bowel disease - ulcerative colitis or Crohn's disease. This measure was 100% accurate in differentiating the two, however it couldn't differentiate between healthy control and the other inflammatory bowel diseases (ischemic colitis, infectious diarrhea, clostridium difficile) with a myeloperoxidase level below 1ug/g. Lactoferrin in healthy patients have extremely low levels in their stool (0.01ug/g), but a cutoff lactoferrin level is less obvious. Average lactoferrin concentrations for inflammatory bowel disease (ulcerative colitis and Crohn's disease) and other inflammatory bowel disease (ischemic colitis, infectious diarrhea and clostridium difficile) patients are 28.91ug/g and 2.23ug/g, respectively. Neither myeloperoxidase or lactoferrin were capable of differentiating types of inflammatory bowel disease within this limited sample size. Results were encouraging and suggest the use of QLISA for myeloperoxidase and lactoferrin analysis to increase differential diagnosis when evaluating patients who may have inflammatory bowel disease. These diagnostic biomarkers can help provide an earlier diagnosis and allow quicker, appropriate treatment, potentially bypassing costly, discomforting diagnostic procedures that are time-consuming for both the patient and the physician.

A quantum dot-based diagnostic immunoassay for biomarker detection in gastrointestinal inflammatory diseases

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A quantum dot-based diagnostic immunoassay for biomarker detection in gastrointestinal inflammatory diseases

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