Biomolecular Detection Assays



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In the present work, three methods for the detection or monitoring of biomolecules are outlined in detail. The first study describes the development of a saliva-based enzymatic screening assay for diabetes mellitus. In the second study, lateral flow assays (LFAs) utilizing commercial colloidal gold and blue latex nanoparticle reporters are compared to persistent luminescence nanoparticle (PLNP; “nanophosphor”) LFAs to assess the sensitivity of nanophosphors as a reporter system. The final study demonstrates the utility of a nanophosphor-based LFA for detecting low concentrations of dengue virus (DENV) biomarker non-structural protein 1 (NS1). 1,5-Anhydroglucitol (AHG) is a naturally occurring monosaccharide and a clinically validated blood biomarker for diabetes. The blood concentration of AHG falls during periods of hyperglycemia, as glucose outcompetes AHG for kidney reuptake. Salivary AHG quantification has been suggested to be useful for diabetes screening but has not been implemented in any widely applicable fashion. We have developed a chemiluminescence assay to quantify AHG in saliva and demonstrated that the assay could distinguish between healthy and diabetic individuals (N = 265; p < 0.0001, ROC AUC = 0.82). These findings suggest that, with further validation, this approach may serve as the basis of a non-invasive tool for diabetes screening. Commercially-available LFAs commonly use colloidal gold or blue latex nanoparticles reporter systems that lack sensitivity and are prone to human error when interpreted visually. We have developed nanophosphors that can detect low levels of antigen. In a comparison study, a nanophosphor-based human immunoglobulin G (IgG) LFA had a limit of detection of 0.625 ng/mL, an 81-fold and 58-fold increase in sensitivity over colloidal gold and blue latex nanoparticles, respectively. Current DENV diagnostic methods are commonly unspecific and cannot detect early infection. We have developed an inexpensive, rapid LFA to detect DENV NS1, a known marker of early dengue infection. Using strontium aluminate nanophosphors as reporters, we achieved a limit of detection of 1 ng/mL DENV serotype 1 NS1 antigen. Our assay is comparable to a laboratory-based NS1 ELISA with a 1 ng/mL limit of detection.



Diabetes mellitus, 1,5-anhydroglucitol, Lateral flow assays, Persistent luminescence, Dengue virus