Oxidized Low-Density Lipoprotein: Preparation, Validation, and Use in Cell Models.

Lipoproteins in plasma are constituted by the least dense chylomicron, very-low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL) that can be separated using commercially available medium such as iodixanol. Iodixanol constitutes the self-generated density gradient to fractionate lipoproteins by rapid ultracentrifugation method, replacing time-consuming protocols. Filling the centrifuge tubes is technically easier and faster than layering salt gradients and is reproducible. The separated lipoproteins by this method are closest to the native state with 80 to 100% recovery possible. Low-density lipoprotein is the major carrier of cholesterol in systemic circulation. The plasma isolated LDL is purified to be used as native LDL and for the preparation of oxidized LDL (oxLDL). The oxLDL is characterized for its oxidation, by various methods based on assay of the lipid and protein oxidation products such as TBARS, conjugated diene formation, and by other methods such as agarose gel electrophoresis. Rapid isolation of LDL particles from human plasma is useful for lipid peroxidation studies, characterization of subclass for functional studies and clinical correlation especially in cardiovascular diseases apart from lipidomic, and proteomic studies. OxLDL preparations are done in vitro chiefly based on copper-induced oxidation; glucose and other prooxidants. Which are used for various studies using animal model and in vitro cell models especially to understand macrophage-mediated atheroma formation, vascular endothelial cell dysfunction, cell signaling studies has scope for extensive research in metabolic dysfunction of various cells.  This chapter deals with one of the applications in the in vitro cell models using macrophage (THP-1 cell line) and human retinal pigment epithelial cell (ARPE-19 cell line) to study the oxLDL uptake using fluorescently labeled oxidized LDL (DiI-oxLDL).

Label-free genosensing of dengue serotypes with an electrodeposited reduced graphene oxide-tris(bipyridine)ruthenium(II).

Constructing a label-free electrochemical transducer platform without compromising inherent biocompatibility against specific bioreceptor remains challenging, particularly probing nucleic acid hybridization at electrode interface without external redox-mediator. Here, we show that electrochemically reduced graphene oxide-tris(bipyridine)ruthenium(II) (ErGO-TBR) nanosheets electrodeposited on carbon screen printed electrode can quantify hybridization of clinically important target sequences specific to serotypes of dengue virus (DENV) non-structural 1 (NS1) protein. Different variables including deposition potential, time, and electrolytic composition were optimized for fabrication of label-free transducer platform. Structural and electrochemical properties of ErGO-TBR/SPE were comprehensively elucidated using microscopic and spectroscopic techniques. Electrochemical quartz crystal microbalance (EQCM) analysis reveals the growth of electrodeposited redox-active species on the electrode interface. Surface functional group investigations suggested that TBR deposited on the basal and edges of ErGO substrate via electrostatic and π-π interactions. Functionalization of bio-affinity layer (B) on ErGO-TBR/SPE enables better loading of probe DNA (PDNA) toward specific detection of DENV target DNA (TDNA) with an ultralow detection limit promising for clinical diagnosis. Scalable chronoamperometry-based redox-active surface growth, customizable bioactivation strategy and external mediator-less probing of nucleic acid hybridization make the present system suitable for other translational application in healthcare diagnosis.