Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay
Single Molecule Protein Detection with Attomolar Sensitivity Using Droplet Digital Enzyme-Linked Immunosorbent Assay
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DOI：
10.1021/acsnano.0c02378
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发表时间：
2020-08-25
期刊：
影响因子：
17.1
通讯作者：
Walt, David R.
中科院分区：
文献类型：
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作者：
Cohen, Limor;Cui, Naiwen;Walt, David R.
许多蛋白质以低浓度存在于生物样品中，因此，超灵敏蛋白质检测技术是必要的。为了克服敏感性的挑战，开发了数字化酶联免疫吸附试验(ELISA)，它比传统的ELISA1000倍灵敏，并允许检测亚毫微克分子蛋白。然而，这种灵敏度仍然不足以测量各种生物样本中的许多蛋白质，从而限制了我们检测和发现生物标志物的能力。为了克服这一局限性，我们开发了液滴数字酶联免疫吸附试验(DdEL ISA)，这是一种使用数字酶联免疫吸附试验和液滴微流控技术检测低蛋白水平的简单方法。DdEL通过提高采样效率和计数更多的靶分子来实现最高的灵敏度。DdELISA可以检测低分子范围的蛋白质，比使用单分子阵列(SIMOA)的数字ELISA法的灵敏度高25倍，单分子阵列是目前超灵敏蛋白质检测的金标准工具。使用ddELISA，我们测量了LINE1/ORF1蛋白，这是一种以前从未在血清中检测到的潜在癌症生物标记物。此外，由于我们设备设计的简单性，ddELSA在医疗保健应用中前景看好。因此，ddELISA将有助于发现以前从未被测量过的各种临床应用的生物标记物。
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.