表面等离子共振（SPR）是一种用于测量结合亲和力和动力学的热门实时技术，台式仪器将该技术的经济性和易用性与其他优点相结合。通过用带有6xHis标签的生物分子配体固定到具有Ni2+-nitrilotriacetic acid（NTA）功能基团的感测表面，可以实现Ni-NTA固定化，尽管Ni-NTA固定化具有许多优点，包括能够再生和重复使用传感器，但使用可能导致实验重复之间的信号变异。我们在这里报道了使用Nicoya OpenSPR作为模型系统研究这种变异性的因素，并提出了一些控制这些因素的方法，以提高数据的重复性和可靠性。我们的模型配体/分析物对是两种卵巢癌生物标志物蛋白（MUC16和HE4）及其相应的单克隆抗体。我们观察到多个NTA芯片上的非特异性结合范围广泛。在相同芯片上运行的实验在配体固定化和分析物结合方面的结果比在不同芯片上运行的实验更加一致。进一步评估表明，不同芯片对同样浓度注入蛋白的最大固定化表现出不同。我们还展示了在高浓度配体下由于立体拥挤引起的配体固定化水平和分析物响应之间的多种关系。通过使用这种校准来指导实验设计，研究人员可以选择与分析物响应线性范围相对应的蛋白浓度进行固定化。我们是第一个通过校准和标准化来增加这些芯片的重复性和数据质量的研究。我们的研究评估了影响芯片变异性的各种因素，填补了有关现有商业可用传感器芯片的知识空白。通过在实验设计过程中控制这些因素，可以最大限度地减小在使用这些重要的传感平台时分析物信号的变异性。

Surface plasmon resonance (SPR) is a popular real-time technique for the measurement of binding affinity and kinetics, and bench-top instruments combine affordability and ease of use with other benefits of the technique. Biomolecular ligands labeled with the 6xHis tag can be immobilized onto sensing surfaces presenting the Ni2+-nitrilotriacetic acid (NTA) functional group. While Ni-NTA immobilization offers many advantages, including the ability to regenerate and reuse the sensors, its use can lead to signal variability between experimental replicates. We report here a study of factors contributing to this variability using the Nicoya OpenSPR as a model system and suggest ways to control for those factors, increasing the reproducibility and rigor of the data. Our model ligand/analyte pairs were two ovarian cancer biomarker proteins (MUC16 and HE4) and their corresponding monoclonal antibodies. We observed a broad range of non-specific binding across multiple NTA chips. Experiments run on the same chips had more consistent results in ligand immobilization and analyte binding than experiments run on different chips. Further assessment showed that different chips demonstrated different maximum immobilizations for the same concentration of injected protein. We also show a variety of relationships between ligand immobilization level and analyte response, which we attribute to steric crowding at high ligand concentrations. Using this calibration to inform experimental design, researchers can choose protein concentrations for immobilization corresponding to the linear range of analyte response. We are the first to demonstrate calibration and normalization as a strategy to increase reproducibility and data quality of these chips. Our study assesses a variety of factors affecting chip variability, addressing a gap in knowledge about commercially available sensor chips. Controlling for these factors in the process of experimental design will minimize variability in analyte signal when using these important sensing platforms.