Xiong Zhen, Potter Colin J, McLeod Euan
Wyant College of Optical Sciences, University of Arizona, 1630 East University Boulevard, Tucson, Arizona 85719, United States.
ACS Sens. 2021 Mar 26;6(3):1208-1217. doi: 10.1021/acssensors.0c02481. Epub 2021 Feb 15.
Accurate, cost-effective, easy-to-use, and point-of-care sensors for protein biomarker levels are important for disease diagnostics. A cost-effective and compact readout approach that has been used for several diagnostic applications is lens-free holographic microscopy, which provides an ultralarge field of view and submicron resolution when it is coupled with pixel super-resolution techniques. Despite its potential as a diagnostic technique, lens-free microscopy has not previously been applied to quantitative protein molecule sensing in solution, which can simplify sensing protocols and ultimately enable measurements of binding kinetics in physiological conditions. Here, we sense interferon-γ (an immune system biomarker) and NeutrAvidin molecules in solution by combining lens-free microscopy with a one-step bead-based agglutination assay, enabled by a custom high-speed light-emitting diode (LED) array and automated image processing routines. We call this a quantitative large-area binding (QLAB) sensor. The high-speed light source provides, for the first time, pixel super-resolved imaging of >10 2 μm beads in solution undergoing Brownian motion, without significant motion blur. The automated image processing routines enable the counting of individual beads and clusters, providing a quantitative sensor readout that depends on both bead and analyte concentrations. Fits to the chemical binding theory are provided. For NeutrAvidin, we find a limit of detection (LOD) of <27 ng/mL (450 pM) and a dynamic range of 2-4 orders of magnitude. For mouse interferon-γ, the LOD is <3 ng/mL (200 pM) and the dynamic range is at least 4 orders of magnitude. The QLAB sensor holds promise for point-of-care applications in low-resource communities and where protocol simplicity is important.
用于检测蛋白质生物标志物水平的准确、经济高效、易于使用且可即时检测的传感器对疾病诊断至关重要。一种已用于多种诊断应用的经济高效且紧凑的读出方法是无透镜全息显微镜，当它与像素超分辨率技术结合时，可提供超大视野和亚微米分辨率。尽管无透镜显微镜作为一种诊断技术具有潜力，但此前尚未应用于溶液中蛋白质分子的定量传感，这可以简化传感方案并最终实现生理条件下结合动力学的测量。在此，我们通过将无透镜显微镜与基于珠子的一步凝集试验相结合，检测溶液中的干扰素-γ（一种免疫系统生物标志物）和中性抗生物素蛋白分子，该试验由定制的高速发光二极管（LED）阵列和自动图像处理程序实现。我们将此称为定量大面积结合（QLAB）传感器。高速光源首次提供了对溶液中经历布朗运动的大于10²μm珠子的像素超分辨成像，且无明显运动模糊。自动图像处理程序能够对单个珠子和簇进行计数，提供取决于珠子和分析物浓度的定量传感器读数。给出了与化学结合理论的拟合。对于中性抗生物素蛋白，我们发现检测限（LOD）<27 ng/mL（450 pM），动态范围为2 - 4个数量级。对于小鼠干扰素-γ，LOD<3 ng/mL（200 pM），动态范围至少为4个数量级。QLAB传感器在资源匮乏社区以及协议简单性很重要的即时检测应用中具有前景。
