SPRi protein arrays for rapid, label-free immunodetection offer an attractive approach for high throughput biosensing and analysis of molecular interactions. However, their routine use for quantitative analysis requires adequate reproducibility and homogeneity of receptor assemblies manufactured using microdispensing techniques. There is also an emerging need to develop versatile and non-destructive methods for the determination of protein surface density for quality control of the immobilization process. Here, we report on a simple, one-step method of surface proteins visualization by their reversible, non-specific staining with anionic dyes: Ponceau S (PS), Amido Black (AB) or Coomassie Brilliant Blue G (CBB-G). Using rabbit IgG antibody and transferrin as model receptors, we demonstrate the possibility of protein surface density determination in a broad range up to ∼ 3 ng∙mm (for a 2D monolayer) and up to at least 12.5 ng∙mm (for a 3D hydrogel-coated substrate). The ranges of dynamic response for the developed methods cover the typical surface densities required for biosensing and studies of the binding kinetics. It was demonstrated that each of the investigated dyes manifests advantages in specified applications. While CBB-G shows the highest sensitivity of SPRi response, AB requires the gentlest labeling conditions, and PS shows the highest association rate and ease of washing out, even from protein layers of high density. The feasibility of the developed method for the quality control of SPRi microarray was confirmed. The advantages of the new, dye-based, non-specific labeling method over immunolabeling in terms of simplicity of implementation and versatility have also been highlighted.

中文翻译：

---

通过多重 SPRi 免疫传感的可逆、非特异性染色来对基于蛋白质的受体层进行质量控制的通用方法


用于快速、无标记免疫检测的 SPRi 蛋白质阵列为高通量生物传感和分子相互作用分析提供了一种有吸引力的方法。然而，它们用于定量分析的常规用途需要使用微分配技术制造的受体组件具有足够的再现性和均质性。人们还迫切需要开发通用且非破坏性的方法来测定蛋白质表面密度，以控制固定过程的质量。在这里，我们报告了一种简单、一步的表面蛋白可视化方法，通过使用阴离子染料进行可逆、非特异性染色：丽春红 S (PS)、氨基黑 (AB) 或考马斯亮蓝 G (CBB-G)。使用兔 IgG 抗体和转铁蛋白作为模型受体，我们证明了在高达 ∼ 3 ng∙mm（对于 2D 单层）和至少 12.5 ng∙mm（对于 3D 水凝胶）的广泛范围内测定蛋白质表面密度的可能性- 涂层基材）。所开发方法的动态响应范围涵盖了生物传感和结合动力学研究所需的典型表面密度。事实证明，所研究的每种染料在特定应用中都表现出优势。虽然 CBB-G 显示了 SPRi 响应的最高灵敏度，但 AB 需要最温和的标记条件，而 PS 显示出最高的关联率和易于洗掉的能力，即使是从高密度的蛋白质层上也是如此。证实了所开发的 SPRi 微阵列质量控制方法的可行性。与免疫标记相比，基于染料的新型非特异性标记方法在实施简单性和多功能性方面的优势也得到了强调。