Single-cell HER2 quantification via instant signal amplification in microdroplets

Abstract

Accurate and ultrasensitive evaluation of human epidermal growth factor receptor 2 (HER2) protein is key to early diagnosis and subtype differentiation of breast cancer. Single-cell analyses to reduce ineffective targeted therapies due to breast cancer heterogeneity and improve patient survival remain challenging. Herein, we reported a novel droplet microfluidic combined with an instant cation exchange signal amplification strategy for quantitative analysis of HER2 protein expression on single cells. In the 160 μm droplets produced by a tapered capillary bundle, abundant Immuno-CdS labeled on HER2-positive cells were replaced by Ag ⁺ to obtain Cd²⁺ that stimulated Rhod-5N fluorescence. This uniformly distributed and instantaneous fluorescence amplification strategy in droplets improves sensitivity and reduces signal fluctuation. Using HER2 modified PS microsphere to simulate single cells, we obtained a linear fitting of HER2-modified concentration and fluorescence intensity in microdroplets with the limit detection of 11.372 pg mL^-1. Moreover, the relative standard deviation (RSD) was 4.2-fold lower than the traditional immunofluorescence technique (2.89% vs 12.21%). The HER2 protein on SK-BR-3 cells encapsulated in droplets was subsequently quantified, ranging from 9862.954 pg mL^-1 and 205.26 pg mL^-1, equivalent to 9.795 × 10⁶ and 2.038 × 10⁵ protein molecules. This detection system provides a universal platform for single-cell sensitive quantitative analysis and contributes to the evaluation of HER2-positive tumors.

Keywords: Droplet microfluidics; HER2 protein expression; Instant cation exchange signal amplification; Quantitative detection; Single-cell.