Single-Molecule Protein Analysis by Centrifugal Droplet Immuno-PCR with Magnetic Nanoparticles

Chuan Zhang, Kaixin Zheng, Chi Li, Ranran Zhang, Yicheng Zhu, Linxiao Xia, Yicheng Ma, Hans M. Wyss, Xiaoyu Cheng (Corresponding author), Sailing He (Corresponding author)

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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.

Original languageEnglish
Pages (from-to)1872-1879
Number of pages8
JournalAnalytical Chemistry
Volume96
Issue number5
Early online date15 Jan 2024
DOIs
Publication statusPublished - 6 Feb 2024

Funding

The authors gratefully acknowledge the Fundamental Research Funds for the Central Universities (Grant nos. 2022LHJH02-04 and K20220146), the National Natural Science Foundation of China (Grant nos. 61905211, 91833303, 62050410447, and 11621101), the “Pioneer” and “Leading Goose” R&D Program of Zhejiang Province (2023C03083 and 2024C03045), the National Key Research and Development Program of China (Grant no. 2022YFC3601000), the Science Foundation of Zhejiang Province (Y21B050009), and the Ningbo Science and Technology Project (No. 2023Z179). The authors thank Prof. Baorong Zhang from the Second Affiliated Hospital of Zhejiang University for providing the antibody, Wei Yin from the Core Facilities, Zhejiang University School of Medicine for technical support, and Julian Evans of Zhejiang University for valuable discussions.

FundersFunder number
Key Research and Development Program of Zhejiang Province2023C03083, 2024C03045
National Natural Science Foundation of China11621101, 62050410447, 91833303, 61905211
Natural Science Foundation of Zhejiang ProvinceY21B050009
National Key Research and Development Program of China2022YFC3601000
Fundamental Research Funds for the Central Universities2022LHJH02-04, K20220146
Science and Technology Project of Nantong City2023Z179

    Keywords

    • Biomarkers/analysis
    • DNA
    • Gold
    • Immunologic Tests
    • Magnetics
    • Magnetite Nanoparticles
    • Metal Nanoparticles

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