Biomarker screening in clinical analysis requires high throughput methodologies to handle the huge amount of samples, selective and sensitive detection methods for being capable of analyzing low abundant biomarkers, simple operational handling steps such as pipetting only, and automatable instrumental analysis steps with high reliability, besides of the fact that the analyzed biomarkers are meaningful and validated.

Our focus is directed to the development of assays for analyzing biomarkers of oxidative stress in human plasma samples. Thereby, we are mainly interested in lipid (per)oxidation and in particular in oxidation of phospholipids. The biomarker of prime interest is oxidized low-density lipoprotein (OxLDL), which is considered as risk factor for atherosclerosis, amongst others. The particular goal of our studies was to develop a screening method for assessment of the oxidative stress level in human serum via measuring the presence of oxidized phospholipids in OxLDL.

The developed method makes use of antibodies (Abs) that were raised against OxLDL and immobilized on gold nanoparticles (GNPs). The resultant anti-OxLDL-Abs modified GNPs are used for selective extraction and enrichment of OxLDL from human plasma samples. After washing, the pellet is treated with methanol to extract (oxidized) phospholipids from the trapped OxLDL while GNP-Ab conjugate. The methanolic supernatant can be used directly to determine the (oxidation) profile of phospholipids in LDL via HPLC-MS/MS or MALDI-TOF.

In this presentation, the systematical study of the bioconjugation chemistry comprising size-controlled synthesis of GNPs, surface functionalization and determination of ligand density will be discussed in detail. Furthermore, the potential for application to analyze OxLDL in plasma samples is briefly outlined.