Hydrothermal Modification of Porous Silica Microbands Improves Single-Cell Assays of the Ether-Lipid Metabolism

Abstract The ability to separate and detect lipid metabolites in single cells has been achieved using a miniaturized thin-layer chromatography (TLC) platform termed picoTLC (pTLC). However, continued innovations in the matrix fabrication, lipid reporter development, and single-cell workflow will broaden the applicability of pTLC to single-cell assays. In this study, we report three advances: a simple post-fabrication process [hydrothermal treatment (HT)] to modify the pTLC silica matrix, a novel clickable lipid reporter to assay metabolism of alkylglycerol (an ether lipid), and a fix-click strategy to prepare cells for the pTLC assay of a metabolized lipid reporter. HT was performed by heating pTLC chips in a silica-saturated aqueous solution with cetyltrimethylammonium bromide (CTAB) followed by slow cooling to generate a co-precipitate of silica/CTAB on the silica matrix. After extraction of CTAB, a mesoporous silica deposition was present with a 3-fold increase in surface area of the silica matrix on the HT processed pTLC chip. The separation performance on pTLC was significantly improved by HT, supporting the separation of five model fluorescent lipids with a resolution >1 for all adjacent lipids that was not achievable without HT. Given the growing importance of ether lipids such as alkylglycerol and its relatives in health and disease, a novel clickable lipid (S)-3-(hexadec-15-yn-1-yloxy)propane-1,2-diol (Reporter 1) was synthesized as a reporter for measuring alkylglycerol metabolism in addition to a companion nonmetabolizable lipid as a control (R)-4-((hexadec-15-yn-1-yloxy)methyl)-2,2-dimethyl-1,3-dioxolane (Reporter-2). A fix-click strategy was adapted to provide a simple, easy-to-use workflow for measurement of the reporter lipid metabolism in single cells by pTLC. These combined advances enabled the identification of metabolites formed from the novel lipid reporters in single leukemic cells and will further broaden the application of pTLC to report lipid metabolism at the single-cell level.