用微小液滴即时信号放大进行单细胞HER2定量分析。
Single-cell HER2 quantification via instant signal amplification in microdroplets.
发表日期：2023 Apr 22
作者：
Xiaoxian Liu, Yifan Zhu, Caoxin Li, Yanyun Fang, Jinna Chen, Fei Xu, Yanqing Lu, Perry Ping Shum, Ying Liu, Guanghui Wang
来源：
Cellular & Molecular Immunology
摘要：
准确和超灵敏地评估人表皮生长因子受体2（HER2）蛋白是早期诊断和乳腺癌亚型鉴别的关键。单细胞分析可以减少由乳腺癌异质性导致的无效靶向治疗，提高患者生存率，但仍具有挑战性。本研究利用微液滴技术和即时阳离子交换信号放大策略，对单个细胞上HER2蛋白的表达进行定量分析。在通过锥形毛细管束制备的160微米液滴中，大量标记在HER2阳性细胞上的免疫-CdS被替换为Ag+，获得刺激Rhod-5N荧光的Cd2+。这种均匀分布和即时放大荧光策略可以提高灵敏度并降低信号波动。通过使用HER2修饰的PS微球模拟单细胞，得出HER2修饰浓度和微滴荧光强度之间的线性拟合，检测限为11.372 pg/mL。此外，相对标准偏差（RSD）比传统免疫荧光技术低4.2倍（2.89％与12.21％）。随后在液滴中封装的SK-BR-3细胞上定量HER2蛋白，范围为9862.954 pg/mL和205.26 pg/mL，相当于9.795 × 106和2.038 × 105个蛋白分子。这个检测系统为单细胞灵敏定量分析提供了通用平台，有助于评估HER2阳性肿瘤。Copyright © 2023 Elsevier B.V. All rights reserved.
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.Copyright © 2023 Elsevier B.V. All rights reserved.