Measuring the binding kinetics of single proteins represents one of the most important and challenging tasks in protein analysis. Here we show that this is possible using a surface plasmon resonance (SPR) scattering technique. SPR is a popular label-free detection technology because of its extraordinary sensitivity, but it has never been used for imaging single proteins. We overcome this limitation by imaging scattering of surface plasmonic waves by proteins. This allows us to image single proteins, measure their sizes and identify them based on their specific binding to antibodies. We further show that it is possible to quantify protein binding kinetics by counting the binding of individual molecules, providing a digital method to measure binding kinetics and analyze heterogeneity of protein behavior. We anticipate that this imaging method will become an important tool for single protein analysis, especially for low volume samples, such as single cells.

测量单个蛋白质的结合动力学是蛋白质分析中最重要和最具挑战性的任务之一。在这里，我们证明使用表面等离振子共振（SPR）散射技术可以做到这一点。SPR由于其非凡的灵敏度而成为一种流行的无标记检测技术，但从未用于单个蛋白质的成像。我们通过对蛋白质在表面等离子体波中的散射进行成像来克服这一限制。这使我们可以对单个蛋白质进行成像，测量其大小并根据其与抗体的特异性结合来鉴定它们。我们进一步表明，可以通过计算单个分子的结合来量化蛋白质结合动力学，从而提供一种测量结合动力学和分析蛋白质行为异质性的数字方法。