Single-Molecule Protein Analysis by Centrifugal Droplet Immuno-PCR with Magnetic Nanoparticles
Chuan Zhang, Kaixin Zheng, Chi Kong Li, Ranran Zhang, Yicheng Zhu, Linxiao Xia, Yicheng Ma, Hans M. Wyss, Xiaoyu Cheng, Sailing He
Abstract
Detecting proteins in ultralow concentrations in complex media is important for many applications but often relies on complicated techniques. Herein, a single-molecule protein analyzer with the potential for high-throughput applications is reported. Gold-coated magnetic nanoparticles with DNA-labeled antibodies were used for target recognition and separation. The immunocomplex was loaded into microdroplets generated with centrifugation. Immuno-PCR amplification of the DNA enabled the quantification of proteins at the level of single molecules. As an example, ultrasensitive detection of α-synuclein, a biomarker for neurodegenerative diseases, is achieved. The limit of detection was determined to be ∼50 aM in buffer and ∼170 aM in serum. The method exhibited high specificity and could be used to analyze post-translational modifications such as protein phosphorylation. This study will inspire wider studies on single-molecule protein detection, especially in disease diagnostics, biomarker discovery, and drug development.