Proximity ligation assays for precise quantification of femtomolar proteins in single cells using self-priming microfluidic dPCR chip

Abstract

The quantification of low concentration proteins can facilitate the discovery of some significant biomarkers, and provide us a more profound understanding of cell heterogeneity when applied to single cell analysis. However, most state-of- art single cell protein detection platforms are bulky, expensive and complicated. Here we report a simple and low cost microfluidic dPCR (digital polymerase chain reaction) chip-based proximity ligation assay (PLA) for the quantification of low concentration proteins. First, standard hCSTB (human cystatin B) protein was used to optimize the related experimental conditions. Comparing to ordinary PLA tests, the results showed that our method achieved femtomolar limit of detection (LOD) with a linear dynamic range over three to four orders of magnitude. Then human CD147 protein, a reported biomarker for hepatoma carcinoma, was detected in single HepG2 and L02 cells. The results showed that there were wide disparities in single cell CD147 abundance for both of the two cell lines. And the average CD147 protein content in single HepG2 cells displayed 2-fold increase in comparison to that in single L02 cells. Comparing to the research findings obtained at bulk level, our method can provide more useful information for diagnosis and targeted therapy of tumors.

Keywords: CD147; Digital PCR; Proximity ligation assay; Single cell; hCSTB.