Abstract
In situ spatial proteomics analysis of a single cell has not been achieved yet, mainly because of insufficient throughput and sensitivity of current techniques. Recent progress on immuno-nucleic acid amplification technology presents tremendous opportunities to address this issue. Here, we report an innovative hybridization chain reaction (HCR) technique that involves computer-aided design (CAD) and reversible assembly. CAD enables highly multiplexed HCR with a sequence database that can work in parallel, while reversible assembly enables the switching of HCR between a working state and a resting state. Thus, CAD-HCR has been successfully adopted for single-cell spatial proteomics analysis. The fluorescence signal of CAD-HCR is comparable with conventional immunofluorescence, and it is positively correlated with the abundance of target proteins, which is beneficial for the visualization of proteins. The method developed here expands the toolbox of single-cell analysis and proteomics studies, as well as the performance and application of HCR.