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Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay.

Many proteins are present at low concentrations in biological samples and therefore, techniques for ultra-sensitive protein detection are necessary. To overcome challenges with sensitivity, the digital enzyme-linked immunosorbent assay (ELISA) was developed, which is 1000X more sensitive than conventional ELISA and allows sub-femtomolar protein detection. However, this sensitivity is still not sufficient to measure many proteins in various biological samples, thereby limiting our ability to detect and discover biomarkers. To overcome this limitation, we developed droplet digital ELISA, a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. Droplet digital ELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. Droplet digital ELISA can detect proteins in the low attomolar range and is up to 25-fold more sensitive than digital ELISA using Single Molecule Arrays (Simoa), the current gold standard tool for ultra-sensitive protein detection. Using droplet digital ELISA, we measured the LINE1/ORF1 protein, a potential cancer biomarker that has not been previously measured in serum. Additionally, due to the simplicity of our device design, droplet digital ELISA is promising for point-of-care applications. Thus, droplet digital ELISA will facilitate discovery of biomarkers that have never been measured before for various clinical applications.