Journal
PROTEOMICS
Volume 11, Issue 24, Pages 4731-4735Publisher
WILEY
DOI: 10.1002/pmic.201100361
Keywords
Antibody microarray; Sandwich immunoassay; Single-molecule detection; Technology; Total internal reflection fluorescence
Funding
- National Cancer Institute [1R21CA151197-01]
- National Institutes of Health [1R01DA025744-01]
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Single-molecule protein analysis provides sensitive protein quantitation with a digital read-out and is promising for studying biological systems and detecting biomarkers clinically. However, current single-molecule platforms rely on the quantification of one protein at a time. Conventional antibody microarrays are scalable to detect many proteins simultaneously, but they rely on less sensitive and less quantitative quantification by the ensemble averaging of fluorescent molecules. Here, we demonstrate a single-molecule protein assay in a microarray format enabled by an ultra-low background surface and single-molecule imaging. The digital read-out provides a highly sensitive, low femtomolar limit of detection and four orders of magnitude of dynamic range through the use of hybrid digital-analog quantification. From crude cell lysate, we measured levels of p53 and MDM2 in parallel, proving the concept of a digital antibody microarray for use in proteomic profiling. We also applied the single-molecule microarray to detect the p53MDM2 protein complex in cell lysate. Our study is promising for development and application of single-molecule protein methods because it represents a technological bridge between single-plex and highly multiplex studies.
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