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

被引:167
作者
Cohen, Limor [1 ,2 ,3 ]
Cui, Naiwen [4 ]
Cai, Yamei [4 ]
Garden, Padric M. [1 ,3 ]
Li, Xiang [1 ,3 ]
Weitz, David A. [1 ,5 ]
Walt, David R. [1 ,2 ,3 ]
机构
[1] Harvard Univ, Wyss Inst Biol Inspired Engn, Boston, MA 02115 USA
[2] Harvard Univ, Dept Chem Biol, Boston, MA 02115 USA
[3] Harvard Med Sch, Brigham & Womens Hosp, Dept Pathol, Boston, MA 02115 USA
[4] Harvard Univ, Sch Engn & Appl Sci, Cambridge, MA 02138 USA
[5] Harvard Univ, Sch Engn & Appl Sci, Boston, MA 02115 USA
基金
美国国家科学基金会; 加拿大自然科学与工程研究理事会;
关键词
single molecule; ultrasensitive detection; immunoassay; biomarkers; clinical diagnostics; droplet microfluidics; PLASMA PROTEOME; AMPLIFICATION; ARRAY; QUANTIFICATION; READOUT; BINDING; PCR; DNA;
D O I
10.1021/acsnano.0c02378
中图分类号
O6 [化学];
学科分类号
0703 ;
摘要
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 immuno-sorbent 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 (ddELISA), a simple approach for detecting low protein levels using digital ELISA and droplet microfluidics. ddELISA achieves maximal sensitivity by improving the sampling efficiency and counting more target molecules. ddELISA 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 ultrasensitive protein detection. Using ddELISA, 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, ddELISA is promising for point-of-care applications. Thus, ddELISA will facilitate the discovery of biomarkers that have never been measured before for various clinical applications.
引用
收藏
页码:9491 / 9501
页数:11
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