Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, United States.
Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts 02115, United States.
J Am Chem Soc. 2020 Jul 15;142(28):12314-12323. doi: 10.1021/jacs.0c04331. Epub 2020 Jun 30.
Measurements of very low levels of biomolecules, including proteins and nucleic acids, remain a critical challenge in many clinical diagnostic applications due to insufficient sensitivity. While digital measurement methods such as Single Molecule Arrays (Simoa), or digital ELISA, have made significant advances in sensitivity, there are still many potential disease biomarkers that exist in accessible biofluids at levels below the detection limits of these techniques. To overcome this barrier, we have developed a simple strategy for single molecule counting, dropcast single molecule assays (dSimoa), that enables more target molecules to be counted through increased sampling efficiency and with a simpler workflow. In this approach, beads are simply dropcast onto a microscope slide and dried into a monolayer film for digital signal readout. The dSimoa platform achieves attomolar limits of detection, with an up to 25-fold improvement in sensitivity over Simoa, the current state of the art for ultrasensitive protein detection. Furthermore, due to its simple readout process and improved cost-effectiveness compared to existing digital bioassays, dSimoa increases amenability to integration into point-of-care platforms. As an illustration of the potential utility of dSimoa, we demonstrate its ability to measure previously undetectable levels of Brachyury, a tissue biomarker for chordoma, in plasma samples. With its significantly enhanced sensitivity and simplicity, dSimoa can pave the way toward the discovery of new biomarkers for early disease diagnosis and improved health outcomes.
由于灵敏度不足，测量包括蛋白质和核酸在内的极低水平生物分子仍然是许多临床诊断应用中的一个关键挑战。虽然数字测量方法，如单分子阵列（Simoa）或数字 ELISA，在灵敏度方面取得了重大进展，但仍有许多潜在的疾病生物标志物存在于可及的生物体液中，其水平低于这些技术的检测限。为了克服这一障碍，我们开发了一种用于单分子计数的简单策略，即滴铸单分子检测（dSimoa），该策略通过提高采样效率和简化工作流程，使更多的目标分子能够被计数。在这种方法中，珠子简单地滴铸在显微镜载玻片上，并干燥成单层膜以进行数字信号读出。dSimoa 平台实现了皮摩尔级别的检测限，与当前最先进的超灵敏蛋白质检测的 Simoa 相比，灵敏度提高了 25 倍。此外，由于其简单的读出过程和与现有数字生物测定相比提高的成本效益，dSimoa 增加了集成到即时护理平台的适用性。为了说明 dSimoa 的潜在应用，我们展示了它在测量先前无法检测到的血浆样本中脊索瘤组织生物标志物 Brachyury 水平方面的能力。dSimoa 具有显著增强的灵敏度和简单性，可以为早期疾病诊断和改善健康结果发现新的生物标志物铺平道路。
