In-Situ imaging detection of cell membrane and intracellular cholesterol via cascade reactions

Herein, an effective membrane-to-intracellular cholesterol detection strategy was designed based on cascade reactions. A biochip array was firstly fabricated by consecutively immobilizing luminol modified gold nanoparticles (Au@luminol), soybean peroxidase (SBP) and cholesterol oxidase (ChoX) on the cellulose acetate (CA) membrane functionalized home-made micropore array. When cholesterol existed, it was oxidized by ChoX generating H₂O₂, which further triggered the CL reaction under the SBP catalysis, the CL signals were collected by a charge-coupled device (CCD). The proposed strategy exhibited a wide linear range from 0.12 μM to 1000 μM and relatively low detection limit (LOD) of 0.08 μM. Furthermore，it could be used to in-situ detect membrane cholesterol and intracelluar esterified cholesterol in HepG2 cells. After activated HepG2 cells were added to the modified biochip, membrane cholesterol was detected directly. Intracelluar esterified cholesterol was detected through the introduction of triton X-100 and cholesteryl esterase (ChoE). Additionally, the cholesterol content in cells was changed after stimulated by drugs, such as apolipoprotein A-I (ApoA-I), pitavastatin or probucol. The correlation of the CL signal with the amount of cholesterol confirmed that our strategy was feasible to simultaneously detect membrane and intracellular cholesterol at different cellular states. The proposed strategy exhibited excellent sensitivity, selectivity, stability, and reproducibility in a simple, cheap way, which opened a new door for studying clinic treatment of the cholesterol-related diseases.