A comprehensive cardiac troponin assay enabled by nanotechnology and top-down proteomics

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 DESCRIPTION (provided by applicant): Heart failure is the leading cause of morbidity and mortality in the United States and is becoming a global health problem. Accurate diagnosis of the disease at the early stage will allow implementation of early intervention strategies that will not only reduce medical costs, but also enable successful treatment of the disease and improve the quality of life. Cardiac troponin I (cTnI), the inhibitory subunit of troponin complex (cTnI-T-), is the gold standard biomarker in the detection of cardiac injuries. However, there are significant challenges in creating a standard and reliable assay using cTnI. One of these challenges is that the commercially available assays for cTnI detection typically use antibodies to target various epitopes of cTnI with varied quality, yielding inconsistent results. Moreover, cTnI typically exist in low abundance but in a myriad forms (phosphorylated, oxidized, degraded, in I-T-C or I-C complex as well as free forms) once released into the blood, which presents a significant challenge for a single antibody-based approach. Herein, I propose to develop a comprehensive cardiac troponin assay enabled by nanotechnology and top-down proteomics. Specifically, I will develop nanoparticles (NPs) functionalized with a range of cTnI affinity ligands to target various epitopes of cTnI to enable the enrichment of the low abundant cTnI proteins from complex blood samples for more accurate detection, effective separation, and subsequent quantitative analysis by high-resolution top-down mass spectrometry. These "artificial antibodies" will eliminate the batch-to-batch variability of the antibodies currently used, allowing for a more reliable assay. Specifically, I will first construct multifunctional superparamagnetic NPs with cTnI binding moieties displayed on the surface. The basic design will consist of iron oxide NPs coated with a silica shell, a fluorescent marker (for detection), cleavable moiety (detachment of cTnI for analysis), and the affinity ligand (for binding with cTnI). After the synthesis of the nanomaterial I will evaluate and demonstrate these functionalized NPs for the enrichment and detection of cTnI from heart tissue, and then blood samples. To rapidly detect the cTnI and related forms, we will develop both Western blot and enzyme-linked immunosorbant assay (ELISA) with these NP-based "artificial antibodies" that contain fluorescent moieties. The captured intact cTnI proteins will be further separated by liquid chromatography and analyzed by high resolution mass spectrometry-based top-down proteomics. The success of the proposed research will help in the development of a more reliable and accurate assay for the detection of cTnI that would result in a better diagnostic of heart disease.